Download IDENTIFICATION OF BACTERIAL PATHOGENS CAUSING

IDENTIFICATION OF BACTERIAL PATHOGENS CAUSING PANICLE
BLIGHT OF RICE IN LOUISIANA
A Thesis
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
In partial fulfillment of the
Requirements for the degree of
Master of Science
In
The Department of Plant Pathology and Crop Physiology
By
Xianglong Yuan
B.S., University of Liaoning, 1985
M.S., Shenyang Agriculture University, 1990
May 2004
ACKNOWLEDGMENTS
I wish to express my sincerest gratitude to Dr. M. C. Rush, my advisor, for
giving me this opportunity to pursue my graduate studies in the Department of Plant
Pathology and Crop Physiology at Louisiana State University. It was Dr. Rush that led
me to enter the world of plant pathology, widening my field of vision in academia. I
sincerely appreciate his guidance, advice, understanding, and patience throughout my
time of study here, particularly in the research work and writing. His working attitude
will necessarily be a beacon lighting my future voyage.
I greatly appreciate Dr. A. K. M. Shahjahan’s help throughout my research
efforts. His generous, patient guidance in my laboratory research, and his many excellent
suggestions made during the past 2.5 years have helped my research efforts greatly.
Special thank go to my committee members, Dr. D. E. Groth, Dr. S. D.
Linscombe, and Dr. J. P. Jones for their suggestions on conducting my research and
writing my thesis. It was their influence that greatly inspired me in developing my thesis.
I acknowledge all of Chinese students that helped me at this university. Their
great and generous support helped me to overcome many difficulties in my life and
during my studies in Baton Rouge, LA.
The most sincere appreciation belongs to my mom and dad. Their encouragement,
love and care were the main source of power that prompted me to accomplish this thesis.
I also express thankfulness to my other family members for their generous support.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................................................................. ii
ABSTRACT............................................................................................................v
CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW .................1
1.1 Disease Distribution and Economic Importance....................................1
1.2 Symptoms and Epidemiology ................................................................3
1.2.1 Symptoms ...............................................................................3
1.2.2 Epidemiology..........................................................................5
1.3 Detection and Identification Methods for Pathogenic Bacteria
on Rice .........................................................................................................7
1.4 Classification, Nomenclature, and Pathological Characteristics
of Pseudomonas and Related Genera .........................................................8
1.4.1 Classification and Nomenclature .............................................8
1.4.2 Characteristics of Burkholderia spp.........................................9
1.4.2.1 B. glumae .....................................................................9
1.4.2.2 B. Plantarii.................................................................10
1.4.2.3 The B. gladioli and B. cepacia Complex ...................10
1.4.2.4 P. fuscovaginae, P. syringae, Acidovorax avenae
( P. avenae) ...........................................................................11
1.5 Virulence..............................................................................................12
1.6 Disease Control....................................................................................13
1.6.1 Chemical Control ..................................................................13
1.6.2 Biological Control.................................................................13
1.6.3 Developing Resistant Cultivars and Lines Using
Genetics and Biotechnology Techniques.......................................13
CHAPTER 2. CLASSIFICATION, IDENTIFICATION, AND
PATHOGENICITY OF BACTERIA ASSOCIATED WITH
PANICLE BLIGHT IN RICE ............................................................................15
2.1 Materials and Methods.........................................................................15
2.1.1 Purification of Isolates .............................................................15
2.1.2 Identification of Isolates Using the BiologTM GN2
Microplate System ............................................................................16
2.1.3 Plant Materials .........................................................................17
2.1.4 Pathogenicity Tests……………………………....... ...............17
2.1.5 Functional Classification of the Identified Isolates .................21
2.2 Results and Discussion .......................................................................22
2.2.1 Purification and Identification of Isolates with the
BiologTM GN2 System ....................................................................22
2.2.2 Pathogenicity Tests and Symptoms Produced .........................38
2.2.3 Colony and Culture Characteristics of the Bacterial
Pathogens ..........................................................................................47
2.2.4 Clustering of Bacterial Strains Isolated from Rice
Showing Panicle Blight into Functional Groups ..............................50
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2.3 Summary and Conclusions ..................................................................56
LITERATURE CITED ......................................................................................61
APPENDIX: BIBLIOGRAPHY OF RELATED LITERATURE...................80
VITA.....................................................................................................................97
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ABSTRACT
Four hundred and two bacterial isolates were isolated on the semi-specific S-PG
medium from diseased rice tissues showing symptoms of panicle blighting. These
isolates were purified using serial dilution in sterile water and replating on S-PG medium.
A total of 420 single isolates were obtained. These isolates were subjected to
pathogenicity tests on rice (Oryza sativa L. cv. Cypress). Based on these tests, 339
isolates were used in BiologTM tests and identified to the species level. Bacterial strains
from 39 species in 16 genera were identified, including 52 isolates representing 15
Pseudomonas species and 261 isolates representing six Burkholderia species. The
remaining 26 isolates included 14 other genera. Of 261 Burkholderia strains, 103 isolates
were B. gladioli, 68 isolates were B. glumae, 60 isolates were B. multivorans, 25 isolates
were B. plantarii, three isolates were B.cocovenenans and three isolates were B.
vietnamiensis. The pathogenicity tests revealed that 69% or 234/339 isolates caused
seedling infection, sheath rot, and/or panicle blighting. Most of the pathogenic strains
were in the genera Burkholderia and Pseudomonas. The four most common species, B.
glumae, B. gladioli, B. multivorans, and B. plantarii, comprised 90% of all of the
pathogenic bacteria, suggesting that a complex of Burkholderia species were causing the
panicle blight/sheath rot syndrome recently found in Louisiana. Five Pseudomonas
species, with total of 16 isolates, were found to be associate with this disease, including
two isolates of P. syringae pv zizanize, three isolates of P. fluorescens, three isolates of P.
pyrrocinia, one isolate of P. spinosa and seven isolates of P. tolaasii. The symptoms of
the disease on rice were only produced by the indicated bacterial strains. Symptoms
included brown margined flag leaf sheath lesions, grain rot, sterile florets, grain
discoloration, and leaf and sheath rot on inoculated plants. It was impossible to
distinguish among Burkholderia species based on symptoms and colony morphology.
Pathogenic Burkholderia strains produced a yellow pigment in King’s B medium.
Avirulent strains did not produce this pigment. The isolates from rice were grouped by
species and pathogenicity. It appeared that B. glumae and B. gladioli were the most
common pathogenic species.
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CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW
1.1 Disease Distribution and Economic Importance
Panicle blighting has been an important sporadic problem in the southern rice
production area of the United States for many years. A somewhat similar disease called
“ear blight”, pecky rice, or grain discoloration has been attributed to fungal causal agents
(Lee, 1992a; Lee, 1992b). This problem is characterized by discoloration of the grain and
panicle branches, usually with distinct lesions, and many fungi have been described as
causing this disease (Atkins, 1974; Ou, 1985; Lee, 1992b). A symptom commonly called
“panicle blight” has been observed for many years in the southern rice producing areas of
the United States. It was considered to be a disorder of undetermined cause as no
pathogen was isolated from diseased tissues (LSU Agricultural Center, 1987; LSU
Agricultural Center, 1999; Groth, et al., 1991). The disease/disorder was characterized as
having panicles with brown or straw-colored discoloration of florets (Groth et al., 1991).
Panicle branches remained green, without lesions or discoloration, and affected florets
stopped developing or aborted. Affected panicles had few to all of the florets diseased.
When the disease was severe the panicle remained upright as the grain did not fill. In
1996 a bacterial pathogen was recognized as the cause of the rice panicle blight syndrome
(Rush, 1998; Shahjahan, 1998, 2000a, 2000b, and 2000c). Rush and Shahjahan revealed
that Burkholderia glumae (formerly Pseudomonas glumae) was the agent causing the
bacterial panicle blight (BPB) disease in Louisiana and adjacent states. Surveying
showed that the BPB occurred in 60 percent of Louisiana fields, suggesting that the
bacterial pathogen was prevalent throughout the rice production areas of the state
(Shahjahan, 2000b).
The world wide distribution of this disease has been become clear as recent
survey reports became available. Studies of the epidemiology, etiology and control of
this disease have attracted the attention of rice plant pathologists from several countries.
According to S.H. Ou (Ou, 1985) bacterial sheath rot and grain blighting on rice was first
reported in Hungary (Klement, 1955). The disease was described as caused by
Pseudomonas oryzicola (later shown by Visnyovszky et al. (1971) to be a synonym of
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Pseudomonas syringae pv. syringae (Ou, 1985). According to Ou (1985), research on
bacterial grain rot was initiated by Goto in Japan (Goto and Ohata, 1956; Goto et
al.,1987). Grain rot in rice was reported to be caused by several bacteria, including
Pseudomonas glumae (Burkholderia glumae), but only P. glumae caused seedling blight
on inoculated plants (Goto et al., 1987). According to Hashioka (1969); Kurita and Tabei,
1967 named the pathogen of grain rot Pseudomonas glumae Kurita et Tabei. The
temperature optimum for growth of P. glumae was reported as 30-35C with a range of
11-40C and a thermal death point at 70C (Kurita et al., 1964). Tanii, et al. (1974)
isolated several bacteria from rice grains showing grain rot. They identified three
Pseudomonas species, but not P. glumae. P. glumae was isolated from plants in nursery
flats with seedlings showing bacterial seedling rot symptoms (Uematsu, et al., 1976).
Since the 1980’s, the disease has been reported from several different countries in
the world, including Korea (Jeong et al., 2003), Taiwan (Chien and Chang, 1987), Latin
America (Zeigler et al., 1987; Zeigler and Alvarez, 1989a; 1989b; 1989c; 1990 and
Zeigler, 1990), Vietnam (Trung et al., 1993), Philippines (Cottyn et al., 1996a; 1996b)
and the Gulf of Mexico rice production area in the U.S. (Rush, et al.,1998; Shahjahan et
al., 1998; 2000b; 2000c). It is reasonable to believe that this disease frequently occurs in
rice around the world, particularly in tropical countries. It is not surprising as the
pathogen is readily seedborne.
Rice is considered to be one of the most important food crops in the world (Lu
and Chang, 1980). It is clear that wide-spread development of a major rice disease would
be disadvantageous to the world’s economy. The direct agronomic consequences of such
an event would be a severe decrease in crop yield and stability of production. BPB has
the potential to reduce the yield of rice as much as 75% in severely affected regions due
to reduction in grain weight, floret sterility, inhibition of seed germination, reduction of
stands, as well as the year-to-year transmission because of the seedborne nature of the
pathogen (Trung, et al., 1993).
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1.2 Symptoms and Epidemiology
1.2.1 Symptoms
The reported symptoms for this disease syndrome were observed in Louisiana on
the leaf sheath of seedlings, flag leaf sheath, and panicle florets. The disease was
described as sheath rotting (brown, necrotic lesion with a distinct margin); leaf stripe;
flag sheath browning (Figure 1); grain rot (Figure 2); spikelet discoloration (grayish or
straw-colored, with bottom half of the floret darkening); sterile florets and/or seedling
blighting ( Shahjahan, 2000b). Several bacterial species have been proposed as causing
similar symptoms. The suspected pathogens are mainly distributed in two taxonomically
and physiologically close genera, Pseudomonas and Burkholderia. B. glumae causes rice
grain rot and seedling rot (Uematsu et al., 1976), sheath rot complex, and grain/sheath rot
(Cottyn et al., 1996a, 1996b, and 2001). The bacterial stripe disease, with grain
discoloration, was reported as caused by Pseudomonas avenae (Kadota and Ohuchi,
1983). Sheath rot and grain discoloration in Latin America was believed to be caused by
Pseudomonas fuscovaginae and Pseudomonas glumae (Zeigler and Alvarez, 1987;
Zeigler and Alvarez, 1989b). However, the different pathogens share similar symptoms
among them. For instance, P. syringae, P. fluorescens and B. glumae caused similar early
symptoms (Goto et al., 1987). Zeigler reported the symptoms produced by Pseudomonas
fuscovaginae as extensive water-soaking and necrosis with poorly defined margins and
glume discoloration before panicles emerge from boots. Infected grain varied from
completely discolored and sterile to nearly symptomless with only small brown spots
(Zeigler and Alvarez, 1987; Zeigler et al., 1987; Zeigler and Alvarez, 1989a; Zeigler and
Alvarez, 1989c).
Symptoms typically caused by B. glumae in Louisiana were panicle blighting with
floret discoloration (with a gray-brown color), usually on the lower half of the developing
grain, with a clear deep brown border followed by sterility or partial filling of the florets
causing the panicles to stand erect (Shahjahan et al., 1998; Shahjahan et al., 2000b). It is
clear that accurate identification of the pathogens and epidemiological surveys are
essential before proposing practical control schemes.
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Figure 1.1. Flag leaf sheath rot, caused by Burkholderia glumae on inoculated Cypress
rice.
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Figure 1.2. Panicle blighting and grain rot, caused by Burkholderia glumae, on inoculated
Cypress rice.
1.2.2 Epidemiology
Bacterial pathogens of plants exist ubiquitously in the plant’s ecosystems and are
usually found in the air, soil, and water. Soil distributions of these pathogens are affected
by soil type, pH value, plants cultivated, and weather conditions. It has been established
that pathogenic bacteria exist on the phylloplanes of rice plants during the growing
season (Matsuda and Sato, 1987; Hikichi, 1993a; Hikichi, 1993b; Hikichi et al., 1993;
Otofuji et al., 1988; Tsushima et al., 1996), on or in rice seeds stored at room temperature
during the winter (Tsushima et al.1987; Tsushimi et al., 1989), on weeds in the field and
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in rice tissues from the previous crop buried in soil (Sogou and Tsuzaki, 1983). The
disease can develop from inoculum in infected seeds from the previous year, in soil, and
on weeds in the field. In the process of infection, host susceptibility, inoculum density,
and climatic factors play the key roles (Tsushima et al., 1985; Tsushima et al., 1987;
Tsushima and Naito, 1991; Tsushima et al., 1995a; Tsushima et al., 1996; Tsushima,
1996).
This disease tends to break out under conditions of unusually high temperatures,
especially at night, and frequent rains (Tushima et al., 1985; Mew, 1992; Zeigler and
Alvarez, 1990). In 1995 and 1998 there were severe incidences of rice BPB in Louisiana
as well as the other Southern rice production areas. Yield losses as high as 40% were
observed in some fields (Shahjahan, 2000b). Record high temperatures were recorded
during these seasons, with high temperatures extending into the night. High humidity
levels at the flowering stages were reported to be particularly conductive to spikelet
infections (Tsushima et al., 1995b). The most susceptible period for floret infections
appears to be during panicle emergence and flowering. Inoculation during flowering
gives the highest rates of floret infection and production of diseased spikelets (Shahjahan
et al., 1998). In these tests using B. glumae, a high level of floret infection continued for
up to 3 days after flowering in inoculation experiments.
The infection rate of the pathogen also depends on inoculum density. Pot
experiments conducted by spraying emerging panicles revealed that infection increased
proportionally as the lognormal volumes of inoculum density (Tsushima et al., 1985).
Based on their experiments, 102 to 104 cfu was proposed as the minimum inoculum
density when spraying in the field and not less than1 cfu/ml was an appropriate dose for
the injection method (Hikichi, et al. 1994).
Pathogen cells present on leaf sheaths play an essential role in primary infection.
Infection on the leaf sheath provides the primary source of inoculum to the emerging
panicle (Tsushima et al., 1991 and Tsushima et al., 1996). We have also observed that
that following injection of the pathogen into boots the first visible diseased tissue was
always on the flag leaf sheath followed by panicle infection. When rice plants are
developing from apparently healthy seeds, the uninfected flag leaf sheath forecasts that
the panicle should be healthy. Therefore, the frequency and severity of infection of the
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flag leaf sheath is an important indicator of the eventual infection on the panicle because
the sheath is spatially close to panicles and the primary time of infection was at heading
time (Tsushima, 1996; Tsushima et al., 1996).
The primary infection site by B. glumae is apparently through the plumules
(Hikichi, 1993a; Hikichi, 1995b). The bacterium enters the lemma and paleae through
stomata, multiplies in intercellular spaces of the parenchyma (Tabei et al, 1989), and
moves towards the surrounding healthy cells and tissues. The bacteria were detected in
the epidermis, parenchyma, and sclerenchyma of glumes of naturally infected rice seeds
using antiserum (Hikichi, 1993b). The long-distance movement of bacteria was
accomplished via vascular systems. Bacteria were observed, by the author (X. Yuan),
flooding out of vascular systems of severely infected rice by tissue sectioning techniques.
1.3 Detection and Identification Methods for Pathogenic Bacteria on Rice
After evaluating the efficacy of the various methods, it appeared important to
select an appropriate combination of methods to recognize the pathogens. A good
identification scheme depends not only on developing a satisfactory resolution level of
methods, but also on the group of bacteria studied (Welch, 1991). Apparently, it is
difficult to differentiate pathogens that are closely related physiologically and
taxonomically by the symptoms they produce and by their growth on selective media. A
semi-selective medium for B. glumae was developed by Tsuschima et al., 1986; however,
other Burkholderia species will also grow on the SP-G medium. Traditional biochemical
and serological methods still have powerful advantages. Cottyn et al., 1996a and 1996b
detected 204 pathogens strains distributed among seven species by using a
comprehensive scheme including antiserum, API 20NE, BiologTM, and cellular fatty acid
methyl ester-fingerprinting. The best differentiation was given by the BiologTM system
(Cottyn et al., 1996b). Although immunological methods possess quiet high resolution,
specific antibodies to some antigens are not readily obtained and this may limit the wide
application of these methods. Recently, PCR-based DNA-typing methods based on both
conservation and variance of rDNA genes among species and pathovars, and other
molecular based techniques, have become popular due to the rapidity, accuracy, and
relative simplicity of operation. These methods will undoubtedly become powerful tools
7
in the field of classification and identification of microorganisms (Furuya et al., 2002;
Karpati and Jonasson, 1996; Mizuno et al., 1995; O’Callaghan et al., 1994; Pelt et al.,
1999; Reiter et al., 2000; Tyler et al., 1995; Ura et al., 1998; Whitby et al., 2000a; Whitby
et al., 2000b.
1.4 Classification, Nomenclature and Pathological Characteristics of Pseudomonas
and Related Genera
1.4.1 Classification and Nomenclature
It has long been known that some species in the genus Pseudomonas were plant
pathogens. According to Burkholder (1949), as early as 1921 Pseudomonas gladioli was
identified as the causal agent of flower rot of gladiolus. Burkholder described a new
bacterial pathogen causing onion sour skin and rots and proposed the bacterium as
Pseudomonas cepacia n. sp. (Burkholder, 1949)., However it was not recognized that
some Pseudomonas spp. could serve as severe human pathogens until the 1960’s.
Recently, it has been confirmed that B. cepacia and B. gladioli can produce human cystic
fibrosis disease (CF) and pulmonary disease (Baxter et al., 1997; Parke and GurianSherman, 1998; Vandamme, 1996, Vandamme et al., 1997, and Vandamme et al., 2000).
The genus Burkholderia was not established until 1992 when Yabuuchi proposed to
transfer seven species previously in the Pseudomonas homology group II to the new
genus Burkholderia (Yabuuchi et al., 1992). P. plantarii and P. glumae were transferred
to this genus in 1994 (Urakami et al., 1994). Yabuuchi et al. reclassified two
Burkholderia species, B. solanacearum and B. pickettii, and Alcaligenes eutrophus, into
the novel genus Ralstonia (Yabuuchi et al., 1995). Until 2002, there were 28 species in
the genus Burkholderia (Table 1.1). Many of the species were believed to be plant
pathogens causing rice, tobacco, maize and other crop diseases. The established rice
pathogens included P. avenae (Goto and Ohata, 1956), P. fuscovaginae (Zeigler, 1987,
1989, 1990; Rott, 1989 and 1991; Cottyn, 1996 and 2001), P. syringae, B. glumae (Goto
and Ohata, 1956; Goto,1965 and Goto and Ohata,1987; Hikichi et al., 1993d, Hikichi et
al.,1994, and HIkichi et al.,1995a; Tsushima et al., 1985, Tsushima et al.,1986, and
Tsushima, 1991; Cottyn et al., 1996a, 1996b and 2001), B. plantarii (Azegami et al.
8
1985, Azegami et al.,1987 and Azegami et al.1988), B. gladioli (Cheng, et al., 2001), and
B. vietnamiensis (Trung et al., 1993).
Table 1.1. Bacterial species described in the genus Burkholderia.*
B. andropogonis
B. anthina
B. ambifaria
B. caledonica
B. caribensis
B. caryophylli
B. cocovenenans
B. fungorum
B. gladioli
B. glathei
B. graminis
B. glumae
B. kururiensis
B. mallei
B. cepacia genomovar I
B. multivorans (formerly genomovar II)
B. cepacia genomovar III
B stabilis (formerly genomovar IV)
B. vietnamiensis (formerly genomovar V)
B. cepacia genomovar VI
B. plantarii
B. pseudomallei
B. phenazinium
B. pyrrocinia
B. sacchari
B. thailandensis
B. ubonensis
B. vandii
* Coenye et al., 2001; Estrada-de los Santos et al., 2001; Gillis et al., 1995; Vandamme,
1996; Vandamme et al.,1997; Vandamme et al., 2000; Yabuuchi et al., 1992.
1.4.2 Characteristics of Burkholderia spp.
1.4.2.1 B. glumae
B. glumae was first recorded as a rice pathogen in the genus Pseudomonas (Goto
and Ohata, 1956), and is considered the major bacterial agent causing seedling and grain
rot in Japan. The typical symptoms were described as sheath brown rot (Cottyn et al.,
1996a and Cottyn et al., 1996b), grain rot and/or discoloration (Cottyn et al., 1996; Goto
and Ohata, 1956 and Goto et al., 1987; Tsushima et al., 1986; Mew et al., 1990; and
Zeigler and Alvarez,1989a.), and seedling rot (Uematsu et al., 1976a and Uematsu et al
1976a; Tsushima et al., 1986). B. glumae is a nonfluorescent bacterium producing a
yellow-green, water-soluble pigment on various media. The organism is rod-shaped, with
1-3 polar flagella. The colony is grayish white or yellow due to the pigment. Arginine
dehydrolase, oxidase reaction and nitrate reduction express a negative reaction.
Lecithinase production and the utilization of L-arginine and inositol are positive.
9
1.4.2.2 B. plantarii
The existence and pathogenicity of B. plantarii was first recognized in northern
rice production areas in Japan (Azegami et al., 1987). The pathogen was found to be
distributed on the weeds in fields and in seed stored at room temperature. B. plantarii is
often isolated when isolating B. glumae, suggesting that they may share a similar
transmission path and life cycle. They also show similar symptoms on rice (Azegami et
al., 1987 and Azegami et al., 1988; Tanaka et al., 1994). However, so far this organism
has only been reported from two countries. Azegami et al., (1987) reported that B.
plantarii produced a reddish-brown pigment on media.
1.4.2.3 The B. gladioli and B. cepacia complex
B. gladioli and B. cepacia are organisms with wide distribution, and have been
isolated from soil, water, plant roots, plant rhizospheres, and the lungs of CF patients
(Holmes et al., 1998; Segonds et al., 1999; Vandamme et al., 1997). For instance, B.
multivorans (B. cepacia genomovar III) was found to be a common plant-associated
bacterium (Balandreau et al., 2001), and can colonize rice, corn, maize, pea, sunflower,
and radish. In natural niches, B. gladioli and B. cepacia regularly coexist with each other,
with B. glumae, and/or occur in the form of hybrids of B. gladioli and B. cepacia. These
hybrids may have a significant pathogenic importance (Baxter et al., 1997). Cheng et al.
(2001) revealed that B. gladioli was one of the causal agents of rice BPB in Louisiana. In
Japan, the symptoms on rice infected with B. gladioli were reported as necrosis or
chlorotic spots on the leaf (Furuya et al., 1997). In addition, necrosis with water-soaked
lesions on tobacco leaves was recorded (Furuya et al., 1997), revealing that this
bacterium has a wide host range.
In addition to being a human pathogen, B. cepacia has been reported to promote
maize growth (Bevivino et al., 1998), to enhance crop yields (Chiarini et al., 1998;
Tabacchioni et al., 1993), to suppress many soilborne plant pathogens (Bevivino et al.,
1998; Hebbar et al., 1998; McLoughlin et al., 1992), and to degrade diverse pesticides
(Daubaras et al., 1996; McLouglin et al., 1992). B. cepacia has demonstrated an effective
role in biological control of soilborne, foliar, and post-harvest plant diseases. However, it
has recently been recognized that this application may pose risks to human health and
10
create environmental problems (Holmes et al., 1998). Aspects of symptomology and
epidemiology on rice concerned with this organism still remained little reported.
1.4.2.4 P. fuscovaginae, P. syringae and Acidovorax avenae (P. avenae)
Another widely observed pathogenic bacterium on rice is the nonfluorescent
bacterium P. fuscovaginae, which produces symptoms similar to those produced by the
previously mentioned bacterial pathogens on rice, and has a worldwide distribution.
Ziegler and Alvarez, (1987a) reported that the bacteria P. fuscovaginae and P. syringae
pv. syringae caused sheath brown rot and grain and sheath discoloration on rice in Latin
America. P. fuscovaginae, P. syringae pv. syringae, A. avenae, and B. glumae were all
identified from rice in Colombia and Central America (Zeigler and Alvarez, 1987b;
Ziegler and Alvarez, 1989a; Ziegler and Alvarez, 1989b; Ziegler, 1990).. Of them, P.
fuscovaginae and A. avenae were the most common pathogens (Zeigler, 1990). Rott et
al., (1989) showed that there were intra-species differences among the native strains of P.
fuscovaginae in Madagascar and reference strains by serological techniques. This
suggested that epidemiology studies and surveys could be complicated by strain
differences.
P. syringae was first detected by Kuwata (1985) on rice in Japan where it caused
the halo blight disease on rice. This new rice bacterial disease had symptoms of small
brown foci surrounded by large, yellow halos on leaf blades (Kuwata, 1985).
The disease on rice caused by A. avenae (formerly P. avenae) shows brown
stripes on leaf blades and sheaths extending over the whole leaf blade in the severest
infections (Ou, 1972; Kodota and Ohuchi, 1983; Shakya and Chung, 1983; Shakya et al.,
1985; Tominaga et al., 1983). The pathogen has a wide host range, causing diseases on
barley, maize, millet, oats, and rice (Kadota, 1996).
The research of Cottyn et al. (1996a and 1996b) demonstrated the existence and
pathogenicity of Philippines strains of P. fuscovaginae, P. syringae pv. syringae, A.
avenae, and B. glumae. However, his reports simultaneously pointed out that none of
these bacteria could be associated with distinct symptoms and B. glumae could not be
differentiated from P. fuscovaginae, P. syringae, and A. avenae based on symptoms,
suggesting that the limitations of this method indicated that a comprehensive detection
scheme and more advanced techniques appeared to be necessary.
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1.5 Virulence
Sato et al. (1989) isolated two bioactive substances from a culture filtrate of B.
glumae, identified as toxoflavin and fervenulin, and indicated that these phytotoxins
could induce chlorotic spots on detached rice leaves. Toxoflavin, a yellow crystalline
solid, first isolated in 1933 from B. cocovenenans (Buckle et al., 1990), reduces the
elongation of sprouts and roots of rice seedlings (Suzuki et al., 1998; Yoneyama et al.
1998). When rice panicles were treated with the substance a brown band on the palea and
lemma, a characteristic symptom of bacterial grain rot, was observed (Liyama et al.,
1995).
This phytotoxin was also detected in B. glumae, several strains of B. gladioli
(Suzuki et al, 1998), and B. cocovenenans, not only from culture filtrates, but also from
rice seedlings infected with the bacteria (Liyama et al. 1994; Liyama et al., 1995; Liyama
et al., 1998). Wang compared the virulent effects of pigment producing strains and nonproducing strains of B. glumae on rice seedlings and potato tuber slices. The results
indicated that all of the 28 pigment producing strains used were virulent to rice seedlings,
whereas all of non-producing strains were avirulent with two exceptions (Wang et al.,
1991). Liyama et al.(1994 and 1995) also demonstrated that virulence to rice was closely
correlated with the toxin after making comparisons between the phytotoxin producing
strains and non-producing strains. By using a transposon mutagenesis technique that
destroys the genes encoding toxin-biosynthesis or virulence-related pathways, transposon
mutants of B. glumae were obtained. The results showed that the non-toxin producing
mutants were unable to induce rice bacterial rot disease (Yoneyama et al., 1998). In
addition, there is a report indicating that the virulence of B. glumae declined rapidly with
continued transfers on PSA medium, suggesting that the loss of virulence occurs from
subculture to a subculture (Tsushima et al., 1991).
The virulence of B. plantarii was associated with production of the phytotoxin
tropolone, a water-soluble substance, which is produced by some Pseudomonas and
Burkholderia spp. including B. glumae. This compound is bioactive to plants, fungi, and
bacteria (Lindberg et al., 1980; Lindberg,1981). This toxin is a non-benzenoid aromatic
compound with a seven-member ring and gives red crystals of ferric tropolone when
12
chelated with iron. It is not clear whether the reddish-brown pigment on B. plantarii
growth media (Azegami et al., 1985 is associated with tropolone production.
1.6 Disease Control
1.6.1 Chemical Control
Many bactericides are able to effectively control or suppress the occurrence of
seedling rot and panicle rot caused by plant pathogenic Burkholderia spp. including
antibiotics, copper, and copper-containing compounds (Katsubi and Takeda, 1998;
Shahjahan et al., 2000b). Oxolinic acid, a synthetic bactericide developed from quinoline
derivatives, inhibits disease development by Gram negative bacteria. This compound was
highly efficacious for the control of this rice disease, either as seed treatments or foliar
sprays. It exhibited both preventive and curative effects (Hikichi, 1993). A study of the
efficacy of oxolinic acid, with FITC-conjugated antibody and fluorescence microscopy,
demonstrated that only 3% of seedlings expressed measurable symptoms after treatment,
compared with 92% of seedlings from untreated seeds (Hikichi, 1995).
1.6.2 Biological Control
The ability of some avirulent strains of Burkholderia to restrict the development
of rice grain rot has been confirmed. Spraying rice panicles with a mixed suspension of a
virulent strain of B. glumae and an avirulent strain of B. gladioli almost completely
controlled the occurrence of the disease in pot and field experiments (Miyagawa and
Takaya, 2000). Similar efficacy was expressed when the seedlings were sprayed with the
avirulent B. gladioli strain prior to inoculation with the B. glumae strain; whereas no
suppression was observed when the order of inoculation was reversed (Miyagawa,
2000a). Similar results were also observed using avirulent strains of B. glumae (Furuya,
et al., 1991) and of B. plantarii (Ohno, et al., 1992), indicating that pre-treatment of seeds
with avirulent strains is an effective biological control method.
1.6.3 Developing Resistant Cultivars and Lines using Genetics and
Biotechnology Techniques
Developing resistant cultivars and lines using genetic and biotechnological means
is one of the major trends in plant disease control. Transforming rice with the oat thionin
gene gave a measurable resistance against grain rot caused by B. plantarii (Iwai, et al.,
13
2002). Due to a high level of accumulation of thionin in cell walls, this transgenic rice
was protected against the attack of the bacterium. The stained and marked bacteria
distributed only on the surface of stomata, whereas, all of the inoculated wild-type rice
Seedlings, developed from seeds germinated after treatment with bacterial suspension,
were wilted and showed characteristic blight symptoms (Iwai, et al., 2002).
Rush revealed that B. cepacia and B. gladioli were common inhabitants of the
phylloplane of rice in Louisiana (Rush et al., 1998). Previous studies showed that B.
glumae (Shahjahan, 2000a and 2000b) and B. gladioli (Cheng, et al, 2001) were the
causal agents of BPB on rice in Louisiana. However, it is still unclear whether other
bacterial pathogens exist that can cause this disease. In this study more than 400 bacterial
isolates obtained from infected rice panicles collected from rice fields in Louisiana, were
used to conduct pathogenicity tests and the cultures were grouped into functional,
biochemically characteristic strains for proper identification with the BiologTM system.
14
CHAPTER 2
CLASSIFICATION, IDENTIFICATION, AND PATHOGENICITY OF BACTERIA
ASSOCIATED WITH BACTERIAL PANICLE BLIGHT IN RICE
Several hundred strains of bacteria isolated from diseased tissues of rice collected
in Louisiana and showing BPB symptoms were provided by M.C. Rush and A.K.M.
Shahjahan for further purification, for identification to species, to conduct pathogenicity
tests to determine which strains were pathogens, and to determine the virulence of
pathogenic isolates. The objectives of this study were to:
1. Replate by serial dilution and streaking all of the primary cultures provided by
Dr. A.K.M. Shahjahan. Obtain single bacterium cultures from the primary
isolates, and preserve these cultures for further study.
2. Identify all single bacterium cultures to species using the Biolog TM GN
MicroPlate System (Jones, 1993).
3. Conduct pathogenicity tests on rice seedlings and panicles in the boot by
injection of bacterial suspensions of all of the identified bacterial cultures..
4. Identify bacterial isolates pathogenic on rice and quantify virulence of the
pathogenic isolates.
2.1 Materials and Methods
2.1.1 Purification of Isolates
Bacterial isolates isolated previously on the selective medium S-PG (Tsushima et
al., 1986), from diseased grains or rice sheaths (Oryza sativa L.) in samples collected
from Louisiana rice production areas, were used in this study. All of the bacterial isolates
were subcultured on King’s Medium B with agar (KBA) (20g protease peptone #3, 1.5g
KH2PO4, 1.5g MgSO4. 7 H2O, 15 g Bacto-agar, and 15 ml glycerol). All isolates were
purified by serial dilution with sterile distilled water and plating on KBA medium. Plates
15
were incubated at 30C for 48 hr. The bacterial colonies on these plates were examined for
their morphological characteristics. Single colonies, with different cultural
characteristics, from each culture plate were touched with a flamed bacteriological loop
and streaked on KBA medium, incubated at 30C for 48 hr, and then stored in
microcentrifuge tubes at -70oC in 30% glycerol. Each isolate was given a culture number.
Figure 2.1.1. Typical colonies of Burkholderia sp. on S-PG
medium.
Bacterial strains were removed from the freezer as needed for identification or
pathogenicity tests, plated on KBA medium, and incubated at 30C for 48 hr. The
development or failure of development of yellow pigment in the medium was noted for
selected B. glumae and B. gladioli isolates for comparison of pigment production with
pathogenicity of these cultures (Figures 2.25, 2.26, and 2.27).
2.1.2 Identification of Isolates Using the BiologTM GN2 Microplate System
Test isolates were removed from the freezer, plated on KBA medium, and
incubated at 30C for 48 hr. Bacteria were subjected to Gram staining tests (Brock et al,
1994). Gram-negative cultures were transferred onto BUG Agar (BUG Agar with 5%
sheep blood), and incubated at 30C for 24hr. Bacterial cells were removed from the
plates with sterile cotton-tipped swabs, suspended in a GN/GP-IF containing 18 to 20 ml
of sterile “gelling” inoculating fluid (0.40% NaCl, 0.03% Pluronic F-689, 0.02% Gellan
Gum). Cell densities were adjusted with a turbidimeter to 52+/-4% transmittance. The
BiologTM GN2 MicroPlate test panels (Biolog, Hayward, CA) were inoculated and
incubated at 30C for 24-48 hr. Results were analyzed with BiologTM GN2 database
16
version 4.20 to determine the identity of each isolate. Data on each isolate was printed
out and kept in ring binders.
2.1.3 Plant Materials
Ten to 15 rice seeds of the variety Cypress were planted in each 15-cm plastic pot
with soil consisting of a mixture of 2:1:1 soil, sand, and peat moss, respectively. The
plants were grown under natural light with a day temperature of 33C – 41C and 75% 95% relative humidity in a greenhouse on the Louisiana State University campus in
Baton Rouge, LA. Fertilizer was applied in the form of 1 tablespoon of slow release
OsmocoteTM with NPK of 19-5-8 for general plant maintenance. After seedlings were
established they were thinned to a population of 5-8 seedlings per plot (Figure 2.1.2).
2.1.4 Pathogenicity Tests
Bacterial inoculum grown on KBA medium incubated at 30C for 48 hr were harvested
with a sterile cotton swab and suspended in a vial containing 0.9ml of sterile distilled
water (ca. 107-108 cfu/ml). All seedling inoculations were by injection with sterile B-D
TM
1ml syringes using B-DTM 23G1 needles. Inoculation preparation and injection of
bacteria into plants was conducted using sterile technique. The person inoculating plants
wore rubber gloves and rubbed his hands in the gloves with 95% ethanol. Three to five
Cypress rice seedlings in the same pot were inoculated on stems before the plants began
tillering. Inoculum consisted of 0.5ml of each bacterial isolate per stem. The inoculated
seedlings were maintained in the greenhouse. After 2 weeks the disease reaction was
determined on each inoculated plant.
Inoculated seedlings were rated with a four category scale. In this scale 0 = no
symptoms produced after inoculation (Figure 2.1.3), + = slight browning around the
injection site, ++ = a brown lesion 1-2 cm in diameter or spreading up and down the stem
from the injection site with a distinct darker brown lesion, +++ = brown lesion spreading
up the stem from the injection site and with the leaf blade on the inner leaf yellowing or
blighted and turning necrotic (Figure 2.1.4). Plants with a “0” rating were listed as NP or
non-pathogenic in the data tables.
17
Figure 2.1.2. Four-leaf-stage Cypress rice seedlings ready for inoculation in pathogenicity tests.
Figure 2.1.3. Inoculated seedling showing “0”or NP reaction.
18
Figure 2.1.4. Inoculated seedlings showing symptoms typical of a +++ rating
(picture courtesy of A.K.M. Shahjahan).
Cypress plants produced as above were fertilized again at the maximum tillering
stage with 1 tablespoon of OsmacoatTM, and inoculated just before panicle emergence
(Figure 2.1.5) with 1.0ml of bacterial suspension injected into the boot using B-DTM 3ml
syringes with B-DTM 23G1 needles. Inoculated panicles were rated 3-4 weeks after
inoculation and emergence with a similar rating where 0 = no signs of disease on the
panicle or florets, + = some browning of the florets with most grain filling normally; ++
= browning on most florets and some grain not filling, lesions formed on sheaths at
inoculation points, +++ = panicles poorly emerging, or fully emerging and florets
discolored and brown with many florets sterile and panicles tending to remain upright
due to lack of grain filling ( Figure 2.1.6). An elongated lesion usually developed on the
flag-leaf sheath at the inoculation point with virulent strains (Figure 2.1.7).
19
Figure 2.1.5. Plants at the boot stage ready for inoculation.
Figure 2.1.6. Typical +++ reaction for panicles inoculated with a virulent
Burkholderia strain.
20
Figure 2.1.7. Flag leaf sheath rot caused by Burkholderia glumae on inoculated
Cypress rice.
All final pathogenicity ratings and evaluations were made during July to October
in the greenhouse with day-time temperatures ranging from 37-43C and relative humidity
ranging from 75-95%. The high temperatures and humidity favored disease development.
Please note that seedling reactions made in the summer of 2002 were not rated with the
four reaction scale, but were assigned a not pathogenic (NP) or pathogenic (P) rating.
2.1.5 Functional Classification of the Identified Isolates
The isolates of the four most common bacteria, B. glumae, B. gladioli, B.
multivorans, and B. plantarii, were clustered into functional groups based on two
clustering systems used independently. One was based on the four unit scale of severity
and the other was a clustering system based upon the similarity index of an isolate to a
typical strain in the BiologTM database. This was done to evaluate the correlation
between the strains and identification accuracy. An isolate was separated into a group of
high-similarity index or a group of low-similarity indexes based on the BiologTM
identification index.. The threshold value of high or low similarity for B. plantarii was
assigned to be 0.6. Considering larger number of isolates in B. glumae, B. multivorans
21
and B. gladioli, a weight was added to the threshold values of each of these species. The
threshold value of high-similarity or low-similarity for B. multivorans and B. glumae was
shifted up to 0.65 and the index was shifted to 0.7 for B. gladioli. By combining both
clustering systems, 8 groups of bacteria in B. glumae, B. gladioli, B. multivorans, and B.
plantarii were produced; that is, high virulence + high similarity index, high virulence +
low similarity index, low virulence + high similarity index, low virulence + low
similarity index, not virulent + high similarity index, not virulent + low similarity index,
moderate virulence + high similarity index, and moderate virulence + low similarity
index. The results of classifications were represented in Tables 2.2.7, 2.2.8, 2.2.9, and
2.2.10. The distribution characteristics of isolates of B. gladioli and B. glumae after such
classifications were illustrated in Figures 2.2.24, 2.2.25, and 2.2.26.
2. 2 Results and Discussion
2.2.1 Purification and Identification of Isolates with the BiologTM GN2 System
Based upon the previous isolation of bacteria using the S-PG semi-selective
medium, a total of 402 bacterial isolates were isolated and further purified using serial
dilution in sterile water. This procedure gave 420 bacterial isolates. All of these isolates
were subjected to pathogenicity tests based on inoculating Cypress seedlings at the fourleaf stage of growth and panicles in the boot just before emergence.
Of these isolates, 339 were identified to the species or pathovar level using the
Biolog
TM
GN2 system (Figures 2.2.1, 2.2.2, 2.2.3, 2.2.4, and 2.2.5). The bacteria
belonged to 39 species distributed among 16 genera (Table 2.2.1). Burkholderia was the
most common genus, giving 262 isolates. One hundred and three isolates were B.
gladioli, 68 isolates were B. glumae, 25 isolates were B. plantarii, 60 isolates were B.
multivorans, 3 isolates were B. cocovenenans, and 3 isolates were B. vietnamiensis. Fifty
two isolates were placed in the genus Pseudomonas; and another 26 isolates were
distributed among 14 other genera (Table 2.2.1)
Pathogenicity tests on the variety Cypress confirmed that the bacteria that were
pathogenic consisted of 234 of 339 strains tested or 65% of the isolates (Table 2.2.2).
Most of the pathogenic strains were in the two genera Burkholderia and Pseudomonas.
The four most commonly isolated species, B. glumae, B. gladioli, B. multivorans, and B.
22
plantarii, comprised 90% of the pathogenic bacterial strains, indicating that a complex of
Burkholderia species may be the causal agents of the BPB and sheath rot disease recently
identified in Louisiana (Table 2.2.2). This is apparently the first report of B. multivorans
and P. tolaasii ( Table 2.2.3) as bacterial pathogens of rice. Also, this is the first time that
B. gladioli and B. plantarii were clearly determined to be pathogens of rice in Louisiana.
Among the B. glumae isolates tested, 91% were pathogenic on Cypress, suggesting that
there is widespread pathogenicity among natural strains of this species (Table 2.2.2).
Three isolates were tentatively identified as B. cocovenenans and three as B.
vietnamiensis. If these are valid identifications, this suggests that these species only
rarely infect rice in Louisiana.
No isolates of P. fuscovaginae, which was previously reported as a causal agent
of grain discoloration and grain rot in Latin America (Zeigler and Alvarez, 1987a and
Ziegler and Alvarez, 1990) and in Africa (Rott et al., 1989 and Rott et al., 1991), was
detected among bacteria isolated from diseased rice in Louisiana. It was evident that most
of the Pseudomonas species, although associated with Burkholderia species in epiphytic
communities on diseased rice tissues, were not the causal agents. Five of 15
Pseudomonas species showed pathogenicity on Cypress rice (Table 2.2.3). Two strains of
P. syringae pv zizanize, isolated from diseased grains, showed pathogenic characteristics
to both rice seedlings and panicles similar to that reported in the literature. P. syringae pv
tagetis isolated from a diseased rice sheath was not pathogenic in inoculation tests. Three
strains of P. pyrrocinia were rice pathogens consistent with previous reports in the
literature (Table 2.2.3) (Rott et al., 1989). Seven strains of Acidovorax avenae, including
two isolates of A. avenae subsp. avenae and five isolates of A. avenae subsp. cattleyae,
were not pathogenic on rice seedlings or panicles although Zeigler (1990) reported A.
avenae as pathogenic in Latin America. These two subspecies were non-pathogens. In
addition, an isolate identified as Vibrio tubiashii by the BiologTM System, caused
intermediate pathogenicity on inoculated rice seedlings in this study. Whether it is a new
plant pathogenic species needs further research.
For most of the bacterial strains tested, isolates were pathogenic to both seedlings
and panicles (Table 2.2.2). However, there were 26 instances among Burkholderia strains
where the strain was pathogenic on panicles, but not on seedlings. Only one strain was
23
pathogenic on seedlings but not on panicles. This suggests that panicles in the boot or
emerging are more susceptible than 4-leaf-stage seedlings.
Figure 2.2.1. BiologTM GN2 Profile of B. gladioli isolate 382gr-1.
Figure 2.2.2. BiologTM GN2 profile of B. multivorans isolate 99sh-2.
24
Figure 2.2.3. Biolog GN2 Profile of P. syringae pv. zizaniae isolate 319gr-4.
Figure 2.2.4. BiologTM GN2 Profile of B. plantarii isolate 260gr-3.
Figure 2.2.5. BiologTM GN2 Profile of B. glumae isolate156sh-1-b.
25
Table 2.2.1. Genera of bacteria isolated from panicle blighted rice plants
collected in Louisiana.
Genera isolated
Number of species in each genus
Burkholderia
6
Pseudomonas
15
Acidovorax
2
Agrobacterium
1
Aquaspirillum
1
Aeromonas
1
Commonas
1
Chryseobacterium
1
Flavimonas
1
Flavobacterium
1
Herbaspirillum
1
Pasteurella
1
Rhizobium
1
Sphingomonas
2
Stennotrophomonas
1
Vibrio
3
Table 2.2.2. The frequency of isolation of Burkholderia species from rice plants showing
symptoms of bacterial panicle blight.*
Frequency of distribution
Number of isolates
of the species among
Species Identified
of each genus
Burkholderia isolates
68
B. glumae
103
B. gladioli
60
B. multivorans
25
B. plantarii
3
B. vietnamiensis
3
B.cocovenenans
* Isolations on SP-G semi-selective medium (Tsushima et al., 1986).
26
26%
39.3%
22.9%
9.5%
1%
1%
Table 2.2.3. Results of BiologTM identification tests and seedling and panicle
inoculation tests to determine pathogenicity of bacterial isolates from bacterial
panicle blight infected rice collected in Louisiana.
Bacterial species
Pathogenicity Tests*
Isolate number
according to
BiologTM
seedling
panicle
99gr-8-a
++
+++
B. glumae
99gr-2-a
+++
+++
B. glumae
98gr-5
+++
+++
B. multivorans
99gr-3-b
++
+++
B. glumae
99gr-4-a
++
+++
B. glumae
6-1
NP
NP
P. resinovorans
98gr-1
NP
NP
B. gladioli
98gr-8-a
NP
NP
P. tolaasii
99sh-2
+
+
B. multivorans
99gr-11
+++
+++
B. glumae
99gr-8-b
NP
No ID
NP
98gr-12-a
++
++
B. glumae
99gr-6-a
+++
++
B. glumae
99sh-6
+++
+++
B. glumae
80-1
+++
+++
B. glumae
98gr-13-b
+++
+
B. gladioli
99sh-9
+
++
B. glumae
32-5
NP
+/NP**
P. putida
99sh-18
++
+++
B. glumae
101gr-2
+
++
B. glumae
99sh-3
+++
+++
B. glumae
99gr-2-b
+++
+++
B. glumae
99gr-4-b
+++
+++
B. glumae
99gr-7-a
+++
+++
B. plantarii
99sh-15-a
+++
+++
B. multivorans
99sh-12
++
++
B. multivorans
101gr-1
+++
++
B. glumae
98gr-6-a
++
++
B. glumae
P. putida biotype
15R-1
NP
A
NP
99sh-16
+++
+++
B. plantarii
99gr-5-b
NP
++
B. multivorans
*NP = not pathogenic, ratings represent degree of virulence when strain was
pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ =
highly virulent.
** NP/+ = NP or + on different inoculated plants.
27
Table 2.2.3. (continued)
Bacterial species according
Pathogenicity Tests*
Isolate number
TM
to Biolog
seedlings
panicles
98gr-6-b
B. glumae
+++
++
99sh-10
B. gladioli
+++
+++
99sh-5
B. glumae
++
+++
34-1
P. putid biotype A
NP
NP
98gr-2
P. putida
NP
NP
98gr-3
B. multivorans
NP
++
99sh-11
B. glumae
+++
+++
99sh-17
B. multivorans
++
+++
98gr-13-a
B. gladioli
NP
+
99gr-1-b
B. multivorans
++
++
99gr-6-b
B. glumae
++
+++
50-9
P. aurantiaca
NP
NP
99gr-1-a
B. glumae
+++
+++
99sh-7
B. gladioli
NP
NP
99sh-15-b
B. multivorans
+++
+++
99gr-9
B. gladioli
++
+++
99gr-4-b
B. gladioli
+++
+++
99gr-3-a
A. dispar
NP
NP
99gr-7-b
B. glumae
++
+++
11sh-5
B. multivorans
NP
NP
35-2
P. putida
NP
NP
25-2
P. aeruginosa
NP
NP
99gr-5-a
B. glumae
++
+
10-4
S. maltophilia
NP
NP
99sh-14
B. glumae
+++
+++
98gr-12-b
B. glumae
++
++
99sh-4-a
B. vietnamiensis
++
+++
99gr-4-b
P. tolaasii
+++
+++
170gr-4
A.Tumefaciens/Radiobacter
NP
NP
191gr-6
B. plantarii
++
++
146sh-3
P. boreopolis
NP
NP
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
28
Table 2.2.3. (continued)
Bacterial species
according to
Isolate number
BiologTM
Pathogenicity Tests*
seedlings
panicles
167gr-1
P. spinosa
+
++
189sh-7
B. multivorans
++
++
192gr-2
B. gladioli
NP
NP
191gr-1
V. fluvialis
NP
NP
189sh-1
A. a. ss avenae
NP
NP
190gr-4
P. aeruginosa
NP
NP
133sh-3
P. resinovorans
NP
NP
191sh-10
B. glumae
NP
+++
127sh-7
P. spinosa
NP
NP
189gr-8
B. glumae
NP
NP
171gr-2
B. glumae
+
++
190gr-1
B. glumae
+
+++
191sh-1
B. multivorans
NP
NP
156sh-1
P. resinovorans
NP
NP
157sh-1
B. multivorans
+
+
158sh-2
P. resinovorans
NP
NP
154sh-1-a
B. plantarii
++
+++
127sh-6
P. spinosa
NP
NP
170sh-1
B. gladioli
+
+/NP*
190gr-2
B. gladioli
++
++
156sh-1-a
P. resinovorans
NP
NP
171gr-1-a
B. gladioli
++
++
191gr-4
B. gladioli
+
+
170gr-3-a
B. gladioli
++
++
190gr-3
B. gladioli
++
++
166gr-6
P. aurantiaca
NP
NP
171gr-3
B. gladioli
++
+++
189gr-9
B. gladioli
++
++
191sh-2
P. corrugata
NP
NP
191sh-12
B. glumae
++
+++
166gr-1
B. gladioli
NP
+++
189gr-4
B. glumae
+++
+++
189gr-2
B. gladioli
++
++
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
29
Table 2.2.3. (continued)
Bacterial species
Isolate number
according to
BiologTM
189sh-6
A. a. ss avenae
147sh-2
A. a. ss cattleyae
190gr-1
B. glumae
167sh-3
H. seropedicae
188gr-1
B. plantarii
188gr-2
B. glumae
192gr-1
B. gladioli
166gr-2
B. gladioli
184sh+gr-2
S. maltophilia
191gr-2
B. plantarii
180gr-2
F. oryzihabitans
154sh-1-b
S. macrogoltabidus
171gr-1-b
B. gladioli
156sh-1-b
B. glumae
170gr-3-b
B. gladioli
#5: 193gr-1
B. multivorans
213sh-2
P. putida biotype B
198gr-2
P. tolaasii
201gr-1
B. multivorans
192gr-7
B. plantarii
206gr-3
B. plantarii
207gr-2
B. multivorans
217gr-1
B. multivorans
210gr-1
P. agarici
202gr-2
B. plantarii
203gr-2
B, multivorans
209gr-2
B. gladioli
201sh-1
B. multivorans
195gr-7
B. plantarii
196gr-4
B. multivorans
216gr-3-a
B. multivorans
197gr-1-a
B. multivorans
216gr-8
B. multivorans
seedlings
NP
NP
+
NP
NP
++
+
++
NP
++
-***
NP
++
+
+++
+
NP
+
NP
++
+
+
++
NP
+
NP
+
NP
+
+
+
++
++
200gr-4
Pathogenicity Tests*
panicles
NP
NP/+**
+++
NP
+
+++
+++
++
NP
+++
NP
NP
++
+
++
+++
NP
+++
+++
++
++
++
+++
NP
++
+++
+++
NP
+++
++
+++
++
+++
+++
+++
P. tolaasii
P.fluorescens
200gr-5
biotype G
NP
+++
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
*** - = no data available
30
Table 2.2.3. (continued)
Bacterial species
Pathogenicity Tests*
Isolate number
according to
BiologTM
seedlings
panicles
208gr-3
B. multivorans
++
+++
196gr-5
B. multivorans
++
+++
210gr-3
B. gladioli
+++
+++
198gr-3
B. multivorans
+
+++
216gr-3-b
B. multivorans
+++
++
209gr-1
B. multivorans
+++
+++
201gr-2
B. gladioli
+
+++
216sh-1
B. gladioli
+
+++
217gr-2
B. multivorans
++
+++
207gr-4
B. gladioli
++
NP
206sh-1
P. fluorescens
+++
++
192gr-5
B. plantarii
+
+++
197gr-2
B. multivorans
++
+++
195gr-11
B. glumae
++
++
197gr-1-b
B. multivorans
+
+++
213gr-1
P. fluorescens
+
+++
195gr-5
B. multivorans
++
+++
216gr-1
B. glumae
++
++
199gr-2
B. multivorans
NP
++
193gr-5
B. multivorans
++
+++
196gr-1
B. multivorans
NP
+++
200gr-3
B. multivorans
NP
+++
216gr-6
B. multivorans
+
+++
217gr-3
B. plantarii
++
++
203gr-1
B. multivorans
NP
++
195gr-6
B. plantarii
NP
+++
201gr-2
B. multivorans
+
+++
P. fluorescens
210gr-4
+
biotype G
+++
200sh-1
B. multivorans
++
+++
#6: 221gr-3
+++
+++
237gr-3
B. gladioli
+++
+++
217sh-1
B. gladioli
+
++
252gr-5-b
B. gladioli
+++
+++
250sh-1
B. gladioli
+++
+++
221sh-1
B. multivorans
NP
++
219sh-4
B. gladioli
+++
+++
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
31
Table 2.2.3. (continued)
Bacterial species according
Pathogenicity Tests*
Isolate number
TM
to Biolog
on seedlings
on panicles
241gr-1
+++
+++
B. gladioli
220gr-1-b
++
+++
B. glumae
250gr-1
NP
++
B. gladioli
249sh-1
+
+
B. gladioli
218sh-2
++
++
B. glumae
222gr-1-a
++
+++
B. gladioli
227gr-2-a
+++
+++
B. gladioli
220gr-2-b
++
++
B. gladioli
250gr-2
++
++
B. plantarii
237gr-1
+++
+++
B. gladioli
227gr-1
+++
+++
B. gladioli
228gr-1
+++
+++
B. gladioli
221gr-1
+++
+++
B. multivorans
252gr-5-a
+++
+++
B. multivorans
222gr-2
P. syringae pv zizanize
++
+++
230sh-1
++
++
P. pyrrocinia
252gr-2
+
++
B. gladioli
237gr-5
NP
NP
B. gadioli
235gr-2
++
++
B. gladioli
218gr-1
+++
++
B. glumae
252gr-1
+++
+++
B. gladioli
227gr-2-b
+++
+++
B. glumae
252gr-7
++
+
B. multivorans
222gr-1-b
+
+
B. glumae
249gr-2
+++
++
B. gladioli
230gr-1
+++
++
B. gladioli
241gr-3
+++
+++
B. gladioli
218sh-1
++
+++
B. gladioli
236gr-1
++
++
B. gladioli
243gr-1
++
++
B. gladioli
249gr-3
++
+++
P. tolaasii
223gr-1
++
++
B. gladioli
219sh-2
P. syringae pv tagetis
NP
++
238gr-3
+++
++
B. gladioli
236gr-3
+++
++
B. gladioli
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
32
Table 2.2.3. (continued)
Isolate number
235gr-1
252gr-4
252gr-2
219sh-1
230sh-2
235sh-1
220gr-1-a
223gr-2
220gr-2-a
218gr-3
243gr-3
226gr-1
237gr-5
#7: 258gr-5
261gr-9
253gr-2
261gr-1
258gr-8
257gr-2
257sh-1
270sh-4
273sh-1
259gr-1
253sh-1
266gr-1
Bacterial species
according to BiologTM
B. gladioli
B. plantarii
B. gladioli
B. gladioli
B. gladioli
B. multivorans
P. pyrrocinia
B. gladioli
B. gladioli
B. plantarii
B. gladioli
B. glumae
B. gladioli
B. multivorans
B. multivorans
A. facilis
B. multivorans
B. glumae
B. gladioli
B. gladioli
B. plantarii
B. multivorans
B. gladioli
B. multivorans
S. macrogoltabidus
Pathogenicity Tests*
seedlings
panicles
++
+ +++
++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++
+++
+
++
+++
+++
+++
+++
NP
NP
+++
++
NP
+/NP
NP
NP
+++
+++
+++
++
++
+++
NP
NP
+++
+++
+++
+++
++
++
+++
+++
NP
++
256sh-2
NP
NP
R. radiobacter
256gr-2
NP
+
B. multivorans
261gr-2
++
+++
B. multivorans
266gr-5
+++
+++
B. multivorans
259gr-4
NP
++
B. gladioli
261gr-5
++
++
V. tubiashii
273sh-2
+
+++
B. multivorans
258gr-4
+
++
B. gladioli
254sh-4
NP
NP
A. avenae. ss cattleyae
266sh-2
+++
+++
P. tolaasii
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
33
Table 2.2.3. (continued)
Bacterial species
Pathogenicity Tests*
TM
according to Biolog
seedlings
panicles
259gr-5
++
++
B. gladioli
261gr-4
+++
+++
B. multivorans
272gr-4
NP
+
B. multivorans
254sh-1
+++
+++
P. tolaasii
260gr-1
+
+++
B. glumae
258gr-1
NP
++
B. multivorans
258gr-5
+++
++
B. multivorans
256sh-3
NP
+++
P. tolaasii
256gr-1
+
++
B. phenazinium
253sh-2
NP
NP
A. a ss cattleyae
255sh-1
+
++
P. tolaasii
269gr-1-a
+++
++
B. gladioli
257sh-2
++
+++
B. glumae
257gr-1
NP
++
C. meningosepticum
254gr-1
+
+++
B. gladioli
267gr-1-b
NP
NP/+
S. sanguinis
273gr-1
+
+++
B. glumae
260gr-3
NP
+
B. plantarii
257gr-2
NP
+++
B. gladioli
254gr-5
++
+++
B. plantarii
254gr-3
NP
NP/+
F. ferrugineum
259gr-3
+
+++
B. plantarii
265gr-1
+
++
B. glumae
270sh-2
NP
+++
B. plantarii
255gr-2
++
+++
B. multivorans
273gr-4
++
+
B. glumae
267gr-2
++
+
B. plantarii
270gr-1
++
+++
P. tolaasii
254gr-2
+++
+++
B. gladioli
269gr-1-b
++
+++
B. glumae
272gr-2
+++
+++
B. multivorans
106sh-11
+++
+++
B. glumae
396gr-2
NP
NP
B. gladioli
363gr-1
NP
+
B. gladioli
407gr-6
+++
+++
B. gladioli
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
Isolate number
34
Table 2.2.3. (continued)
Bacterial species
Pathogenicity Tests*
according to
Bacterial isolates
BiologTM
seedlings
panicles
387gr-2-b
B. gladioli
+++
+++
406gr-3
B. gladioli
+++
+++
408gr-4
B. cocovenenans
+++
+++
357-8
B. gladioli
++
++
366gr-1
B. multivorans
+++
+++
379gr-1-a-(1)
B. glumae
+++
+++
106sh-7
B. vietnamiensis
+
++
378g-3
B. gladioli
++
+
106sh-9
B. plantarii
NP
NP
357gr-1
B. gladioli
NP
NP
106sh-5
B. glumae
NP
NP
384gr-1
P. viridilivida
NP
NP
395-2
B. gladioli
NP
NP
408gr-7
B. gladioli
+
++
403gr-2
B. glumae
++
+++
980007-1(5)
B. glumae
+++
+++
405gr-4
B. cocovenenans
+++
+++
961149-4(4)
B. gladioli
NP
NP
407gr-3
B. plantarii
+++
+++
357-3
B. gladioli
++
+
106gr-3
B. glumae
++
+
106sh-4-b
B. glumae
+++
+++
387gr-2-a
B. glumae
++
+++
367gr-1
B. glumae
++
+++
395gr-2
B. gladioli
+++
+++
367gr-3
B. glumae
++
+++
254gr-2
B. multivorans
+++
+++
255gr-2
B. glumae
++
++
373gr-1
B. gladioli
+++
+++
318gr-4
B. glumae
++
+++
366gr-2
B. gladioli
NP
NP
318gr-1
B. multivorans
+++
+++
379gr-1-b
B. gladioli
NP
NP
980007-1(6)
B. gladioli
+++
+++
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
35
Table 2.2.3. (continued)
Bacterial species
Pathogenicity Tests*
TM
according to Biolog
seedlings
panicles
382gr-1
B. gladioli
+++
++
385gr-1
B. gladioli
++
+
106sh-4-a
B. glumae
NP
NP
366gr-6
B. vietnamiensis
++
++
398gr-3
B. glumae
+++
+++
373gr-2
P. viridilivida
NP
NP
398gr-1
B. glumae
+++
+++
407gr-8
B. gladioli
+
++
407gr-1
B. gladioli
+++
+++
379gr--1-a-(2)
B. gladioli
++
++
951886-4(3)
B. glumae
++
+++
372gr-1
B. gladioli
++
+++
379gr-1-a-(1)
B. multivorans
++
++
393gr-7
P. tolaasii
NP/+**
321gr-9
B. gladioli
+
+
321gr-2-a
B. gladioli
+++
+
321gr-3
B. gladioli
++
++
951886-4(3)
B. gladioli
+++
++
321gr-6
B. gladioli
++
+
325gr-1
B. gladioli
+
+
321gr-8-a
B. gladioli
++
+
324gr-2
B. gladioli
++
+++
324gr-3
B. gladioli
++
+++
319gr-4
P. syringae pv zizanize
++
+
321gr-2-b
B. gladioli
++
++
321gr-4
B. plantarii
++
++
325gr-2
B. gladioli
++
++
321gr-8-b
B. gladioli
++
++
325gr-4
B. gladioli
++
+++
321gr-7
B. gladioli
++
++
319gr-1
B. gladioli
+++
+++
319gr-6
- ***
+
+
154sh-1-a
B. gladioli
+
++
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
*** - = no data available
Isolate number
36
Table 2.2.3. (continued)
Bacterial species
Pathogenicity Test*
TM
according to Biolog
seedling
panicle
113sh2-7
P
+++
B. glumae
113sh2-4
P
++
B. plantarii
113sh2-5
P
+
B. glumae
113sh2-2-a
P
+
B. glumae
113sh2-8
P
+++
B. glumae
117sh1-3
P
+++
B. glumae
117sh1-8
P
+++
B. glumae
117sh2-3
P
+++
B. glumae
117g1-7-a
NP
NP
B. glumae
116g1-1
P
+++
B. glumae
117g1-8
P
+++
B. glumae
117g1-9
P
+++
B. glumae
117sh1-1
NP
NP
S. maltophilia
120g1-1
P
++
B. glumae
119g1-5
P
+++
B. glumae
123sh-3
118sh-6
NP
NP
A. avenae ss cattleyae
119g1-4
NP
++
B. glumae
119g1-3
P
+++
P. pyrrocinia
120g1-2
P
+++
B. glumae
122sh-1
NP
NP
117g1-11
P
+++
B. glumae
118sh-5
P. group2 (B.-like)
NP
NP
114sh1-4
P
NP
B. cepacia
122sh-5
A. jandaei DNA group 9
NP
NP
113sh2-9
P
+++
B. glumae
113sh2-3
P
NP
B. plantarii
113sh1-10
NP
NP
C. acidovorans
124sh-1
124sh-4
113sh2-2-b
P
+
117g1-7-b
P
++
*NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic
with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent.
** NP/+ = NP or + on different inoculated plants.
Isolate number
37
Table 2.2.4. The frequency of pathogenic bacterial strains of each species
isolated from rice based on inoculation of Cypress rice plants.
Number of
Relative
Total number of
Pathogenic
pathogenic
frequency
isolates of each
bacterial
isolates of each
(%)
species
species
identified species
Burkholderia spp.
62
68
B. glumae
91
B. gladioli
85
103
83
B. multivorans
44
60
73
B. plantarii
20
25
80
B. cocovenenans
3
3
100
B. vietnamiensis
3
3
100
Pseudomonas spp.
P. syringae pv zizanize
2
2
100
P. fluorescens (biotype G)*
3
4
75
P. tolaasii
7
12
58
P. pyrrocinia
3
3
100
P. spinosa
1
3
33
Vibrio spp.
V. tubiashii
1
1
100
* P. fluorescens or P. fluorescens biotype G
2.2.2 Pathogenicity Tests and Symptoms Produced
Pathogenicity tests were applied using the method of injection of bacterial
suspensions into the stems of 4-leaf-stage rice seedlings 1 – 2 days before tillering or into
boots just before panicles emerged using the variety Cypress. Continuous observations
and records for the progressive symptoms were made with a digital camera and notes
were taken for 20 – 25 days after inoculation. A range of leaf and leaf sheath symptoms
was observed on seedlings (Figures 2.2.6, 2.2.7, 2.2.8, and 2.2.9) and the boot leaf sheath
( Figure 2.2.11, 2.2.18, and 2.2.20), emerging panicles including poor panicle emergence
( Figure 2.2.10) grain discoloration, sterile spikelets, and complete blacking of hulls
(Figure 2.2.19). Panicle damage and grain discoloration existed in various degrees of
severity. In the severest case, a poor or total lack of panicle emergence occurred. The
lesions on sheaths developed longitudinally into with brown/black necrosis on sheaths,
with grayish-white centers and dark brown, distinct margins. Symptoms produced by
identified B. glumae cultures were similar among all pathogenic Burkholderia species. In
infections with intermediate virulence, a restricted foci around the injection point was
38
formed on the sheath (Figure 2.2.8). For inoculated rice seedlings, a widespread
symptom was yellow or light-brown rot of the leaf sheath. The development of the
disease depended strongly on weather conditions. Cooler temperatures of 23oC – 25oC
significantly reduced lesion development on affected leaf sheaths. No infection was
expressed as a yellow or small light brown area around the injection site (Figure 2.1.3).
An experiment where seedlings and emerging panicles were simultaneously inoculated
with the same bacterial strains under, under cool temperature conditions, confirmed that
unlike rice seedlings, emerged panicles were still severely damaged. In a few cases, B.
plantarii and B. cocovenenans expressed distinct symptoms. A brown leaf stripe along
mid-veins was produced by B. plantarii (Figure 2.2.7) and with B. cocovenenans leaf
discoloration spread along the leaf blade edge to the tip of the leaf (Figure 2.2.9).
However, whether symptoms can be used to distinguish pathogenic strains needs further
study. Typical infection by virulent or moderately virulent strains of several species of
Burkholderia are shown in Figures 2.2.12 to 2.2.17.
Figure 2.2.6. Leaf sheath rot on Cypress seedlings caused by B. glumae isolate106sh-4-b on the
left and B. gladioli isolate 980007-1(6) on the right. Please note that the whole inoculated leaf
sheath rots when the bacterial isolate is highly virulent.
39
Figure 2.2.7. Brown and tan leaf stripes or lesions on seedling leaf blades infected by B. glumae
isolate 106sh-11on the left and B. plantarii isolate 407gr-3 on the right.
Figure 2.2.8. Low virulence reaction produced by B. gladioli (isolate 357gr-1 on the left) and B.
gladioli (isolate 406gr-3 on the right). Symptoms were expressed as a brown lesion of less than
2cm length around the inoculation site with a distinct darker brown margin.
40
Figure 2.2.9. Leaf discoloration extending along the edge of the leaf blade down to the leaf tip
caused by B. cocovenenans (isolate 405gr-4 on the left) and the uninoculated Cypress control on
the right.
Figure 2.2.10. Poor emergence of panicle with spilelet sterility by caused by B. multivorans
isolate 366gr-1on the left and a dark brown sheath and grain Rot caused by B. glumae isolate
367gr-3 on the right.
41
Figure 2.2.11. Extended brown necrotic lesion caused by a highly virulent strain of B. glumae
(367gr-3 on the left) and a dark brown lesion surrounding the inoculation site on the sheath
caused by the moderately virulent B. glumae strain (398gr-3 on the right).
Figure 2.2.12. Strain 98gr-5 of Burkholderia multivorans showing a virulent (+++)
reaction on Cypress rice after seedling inoculation.
42
Figure 2.2.13. Strain 98gr-5 of Burkholderia tolaasii, on Cypress rice showing
a highly virulent (+++) reaction after seedling inoculation.
Figure 2.2.14. Seedling disease reaction (+++) caused by bacterial strain
99gr-2-b of Burkholderia glumae on Cypress rice.
43
Figure 2.2.15. Highly virulent seedling disease reaction (+++) of bacterial
strain 99sh-10, Burkholderia gladioli on Cypress rice.
Figure 2.2.16. Highly virulent seedling disease reaction (+++) of bacterial
Strain 99sh-16, Burkholderia plantarii on Cypress rice.
44
Figure 2.2.17. Seedling disease ++ reaction of bacterial strain 99sh-4-a,
Burkholderia vietnamiensis on Cypress rice.
Figure 2.2.18. Highly virulent panicle and flag leaf sheath reaction (+++)
on isolate 210gr-3 of Burkholderia gladioli on Cypress rice after boot
inoculation (overview).
45
Figure 2.2.19. Close-up view of severe panicle blighting symptoms on Cypress rice resulting
from inoculation with isolate 210gr-3 of Burkholderia gladioli.
46
Figure 2.2.20. Close-up view of flag leaf sheath lesions produced when Cypress rice boots were
inoculated with isolate 210gr-3 of Burkholderia gladioli.
2.2.3 Colony and Culture Characteristics of the Bacterial Pathogens
Examinations of colony morphology showed that the Burkholderia species could
not be distinguished simply by colony or culture characters. All of the species formed
convex colonies with smooth margins or flat colonies with pleated margins on KBA
medium, releasing yellow pigment or no pigment into the medium. Most cultures had
gray-white or cream-white colonies. In a very few cultures there was a reddish tint to the
colonies. Yellow or gray-white colonies were the most common. B. gladioli colonies
were yellow, gray-white, or yellow-green in color, but all of these colony-types were
pathogenic to rice plants, suggesting that there may be toxins other than the phytotoxin
associated with the yellow pigment. All avirulent colonies of B. glumae, B. gladioli, B.
multivorans and B. plantarii were gray-white or cream white colonies that did not release
47
pigments into the medium (Figures 2.2.21, 2.2.22, and 2.2.23). When selected cultures of
B. glumae and B. gladioli were grown on King’s B medium and observed for production
of pigment (toxin) in the medium, it was clear that cultures that produced the yellow or
green pigments were pathogenic and virulent (Table 2.2.5).
Cultures that did not produce pigment were avirulent. Several non-pathogenic and
non-pigment producing cultures of both B. glumae and B. gladioli were identified in this
experiment. These cultures will be useful for future experiments to determine what
controls toxin production and if this character can be transferred from virulent cultures to
avirulent cultures. It’s possible that the gene(s) for toxin production are carried by a
plasmid that can be transferred among Burkholderia spp.
Figure 2.2.21. Cultures of B. plantarii, virulent strain (left) and avirulent strain (right). Note the
yellow toxin in the virulent culture and the lack of yellow toxin in the medium of the avirulent
strain.
48
Table 2.2.5. Production of yellow or green pigment (toxin) in King’s B medium by
pathogenic and nonpathogenic Burkholderia glumae and Burkholderia gladioli isolates.
Panicle
Seedling
Pigment
pathogenicity
Isolate Number
Bacterial species
pathogenicity
(toxin)*
rating
rating
357gr-1
B. gladioli
NP
NP
N
106sh-5
B. glumae
NP
NP
N
395-2
B. glumae
NP
NP
N
366gr-2
B. gladioli
NP
NP
N
237gr-5
B. gladioli
NP
NP
N
99sh-7
B. gladioli
NP
NP
N
257sh-1
B. gladioli
NP
NP
N
321gr-9
B. gladioli
+
+
W
379gr-1-b
B. gladioli
NP
NP
N
396gr-2
B. gladioli
NP
NP
N
98gr-13-a
B. gladioli
NP
+
N
259gr-4
B. gladioli
NP
++
W
363gr-1
B. gladioli
NP
+
N
189gr-8
B. glumae
NP
NP
W
191sh-10
B. glumae
NP
+++
W
250gr-1
B. gladioli
NP
++
W
321gr-9
B. gladioli
+
+
W
192gr-2
B. gladioli
NP
NP
W
166gr-1
B. gladioli
NP
+++
IM
98gr-1
B. gladioli
NP
NP
N
106sh-4-a
B. glumae
NP
NP
N
319gr-1
B. gladioli
+++
+++
G
398gr-1
B. glumae
+++
+++
I
106sh-11
B. glumae
+++
+++
I
980007-1(5)
B. glumae
+++
+++
I
321gr-2-a
B. gladioli
+++
+
IM
406gr-3
B. gladioli
+++
+++
I
382gr-1
B. gladioli
+++
++
IM
407gr-1
B. gladioli
+++
+++
I
99gr-6-a
B. glumae
+++
++
I
99gr-2-a
B. glumae
+++
+++
I
* Production of pigment (toxin) is indicated as “I” = intense, “IM” = intermediate, “W” =
weak (very faint color produced), and “N” = none. “G” = no yellow pigment produced,
but green pigment was produced.
49
Figure 2.2.22. Cultures of Burkholderia glumae with a virulent strain on the left and an
avirulent strain on the right. Note the lack of toxin in the medium on the right.
Figure 2.2.23. Cultures of Burkholderia gladioli, virulent strain (left) and avirulent strain (right).
Note the lack of yellow pigment (toxin) in the medium on the right and the presence of yellow
pigment on the left.
2.2.4. Clustering of Bacterial Strains Isolated from Rice Showing Panicle Blight into
Functional Groups
In order to compare the similarity of strains of the different species to
representative strains stored in the BiologTM GN database, bacterial isolates were
clustered into functional groups based upon data from BiologTM identification tests and
pathogenicity tests. Each isolate was therefore assigned two characteristic parameters: a
virulence parameter and a typicality or similarity parameter. Evaluation using the
50
virulence system produced four groups: non-virulent strains, low virulence strains,
middle-virulent strains, and high virulence strains. Evaluation using the similarity system
formed two groups: low similarity strains and high similarity strains (Table 2.2.6). The
threshold value of high / low similarity of B. plantarii was assigned at 0.6. Considering
the larger number of strains in B. glumae, B. multivorans and B. gladioli, a weight was
added to the threshold values; shifting the values of B. multivorans (Table 2.2.7) and B.
glumae to 0.65 and B. gladioli strains were shifted to 0.7.
The results indicated that B. glumae (Table 2.2.9) and B. gladioli (Table 2.2.8)
possessed more virulent strains and higher similarity indexes as strain populations than
that of B. plantarii, suggesting these two species may be the main pathogens in this study.
A higher similarity index implied higher detection accuracy and high similarity to
reference strains. However, low similarity might not mean low detection accuracy, but
imply that they were different types, considering that bacterial metabolic types are
variable (Jones, 1993).
B. multivorans expressed an intermediate pattern of population distribution
between B. plantarii, B. gladioli, and B. glumae according to the above evaluation
system, suggesting that they took second place in the importance of pathogens (Table
2.2.7).
Table 2.2.6. Clustering of Burkholderia plantarii into functional and typical strains.
High Virulence*
Low Virulence
Non-Virulent
Mid Virulence
High
Low
High
Low
High
Low
High
Low
similarity** similarity similarity similarity similarity similarity similarity similarity
407gr-3
270sh-4
99sh-16
99gr-7-a
195gr-7
206gr-3
260gr-3
195gr-6
217gr-3
192gr-5
202gr-2
188gr-1
270sh-2
252gr-4
218gr-3
259gr-3
321gr-4
191gr-2
192gr-7
154sh-1a
217gr-3
254gr-5
267gr-2
191gr-6
250gr-2
* The three-scale system of disease severity was determined by pathogenicity tests.
** A threshold value of similarity to typical strains was assigned with a similarity index
of 0.6 in this table. The similarity was considered as high similarity if the index was more
than 0.6; otherwise as low similarity.
51
Table 2.2.7. Clustering of Burkholderia multivorans into functional and typical strains.*
High Virulence
Low Virulence
Non-Virulent
Mid Virulence
High
Low
High
Low
High
Low
High
Low
similarity similarity similarity similarity similarity similarity similarity similarity
98gr-5
261gr-4
261gr-1
273sh-1
252gr-5a
216gr-3b
209gr-1
254gr-2
258gr-5
221gr-1
366gr-1
318gr-1
99sh-15a
266gr-5
198gr-3
201gr-2
197gr-1b
216gr-3a
99sh-2
273sh-2
258gr-1
261g-9
11sh-5
99gr-5-b
252gr-7
216gr-8
197gr-1a
99gr-1-b
99sh-17
157sh-1
221sh-1
272gr-4
196gr-4
201sh-1
256gr-2
196gr-5
261gr-2
99sh-12
253sh-1
193gr-1
203gr-1
191sh-1
208gr-3
255gr-2
272gr-2
207gr-2
196gr-1
201gr-1
379gr-1
200sh-1
235sh-1
216gr-6
200gr-3
199gr-2
203gr-2
a(1)
217gr-1
217gr-2
197gr-2
195gr-5
193gr-5
* A threshold value of similarity to typical strains was assigned with a similarity index of 0.65 in this table
52
Table 2.2.8. Clustering of Burkholderia gladioli into functional and typical strains.*
High Virulence
High
similarity
Low
similarity
321gr-2a
319gr-1
170gr-3b
243gr-3
241gr-3
249gr-2
237gr-3
237gr-1
227gr-2a
252gr-1
230sh-2
250sh-1
230gr-1
99sh-10
98gr-13b
254gr-2
241gr-1
219sh-4
252gr-2
252gr-5b
236gr-3
Low Virulence
Non-Virulence
Mid Virulence
High
similarity
Low
similarity
High
similarity
Low
similarity
High
similarity
Low
similarity
325gr-1
408gr-7
321gr-9
379gr-1b
321gr-6
99gr-9
191gr-4
192gr-1
166gr-1
99sh-7
325gr-4
220gr-2b
170sh-1
209gr-2
237gr-5
98gr-1
321gr-3
235gr-1
217sh-1
216sh-1
357gr-1
325gr-2
243gr-1
324gr-2
324gr-3
321gr-8a
321gr-8b
321gr-2b
321gr-7
171gr-1b
171gr-3
219sh-1
166gr-2
249sh-1
258gr-4
363gr-1
396gr-2
98gr-13a
257sh-1
257gr-2
254gr-1
366gr-2
192gr-2
407gr-8
250gr-1
154sh-1a
259gr-4
228gr-1
210gr-3
227gr-1
220gr-2a
207gr-4
170gr-3a
235gr-2
99gr-4-a
189gr-2
269gr-1a
238gr-3
382gr-1
9800071(6)
407gr-6
406gr-3
190gr-2
190gr-3
189gr-9
171gr-1a
223gr-1
223gr-2
222gr-1a
407gr-1
395gr-2
387gr-2b
236gr-1
218sh-1
259gr-5
259gr-1
372gr-1
378gr-3
385gr-1
379gr-1a(2)
357-8
357-3
*A threshold value of similarity to typical strains was assigned with a similarity index of 0.70 in this table.
53
Table 2.2.9. Clustering of Burkholderia glumae into functional and typical strains.*
High Virulence
Low Virulence
Non-Virulent
Mid Virulence
High
Low
High
Low
High
Low
High
Low
similarity similarity similarity similarity similarity similarity similarity similarity
189gr-4
252gr-5a
171gr-2
156sh-1b
189gr-8
218gr-1
258gr-8
222gr-1b
265gr-1
191sh-10
188gr-2
226gr-1
99sh-6
260gr-1
99sh-9
106sh-4a
218sh-2
227gr-2b
99gr-11
273gr-1
101gr-2
395-2
220gr-1b
80-1
98gr-6-b
190gr-1
106sh-5
191gr-2
257sh-2
216gr-1
195gr-11
189sh-7
273gr-4
98gr-12b
106sh-4b
398gr-3
269gr-1b
99gr-8-a
99gr-4-b
99gr-3-b
99sh-5
99gr-5-a
367gr-1
99gr-2-b
98gr-6-a
951886
-4(3)
99sh-3
99gr-6-b
98gr-12a
318gr-4
101gr-1
99gr-6-a
99gr-2-a
99sh-14
99sh-11
99gr-1-a
99sh-15b
398gr-1
9800071(5)
99gr-7-b
99sh-18
367gr-3
403gr-2
255gr-2
387gr-2a
106sh-11
387gr-2106gr-3
b
* A threshold value of similarity to typical strains was assigned with a similarity index of 0.65 in this table.
Based on the percentage of isolates of each Burkholderia species among the
bacterial isolates from rice showing BPB symptoms, B. gladioli, B. glumae, and B.
multivorans appear to be the most important species causing BPB in Louisiana rice
(Table 2.2.24).
54
120
The Number of Isolates
100
Relative Frequency (%)
80
60
40
20
n
pl s
an
ie
ta
B. tnam r i i
co
c o ie n
s
ve
ne i s
na
ns
li
ra
io
B.
v
B.
i vo
ult
gl
ad
B.
B.
m
B.
gl
um
ae
0
18
16
14
12
10
8
6
4
2
0
H
ig
hvi
ru
+H
H
ig
ig
hhsi
vi
m
ru
i
+L
Lo
ow
w
-s
-v
im
iru
i
+H
Lo
ig
hw
si
-v
m
iru
i
+L
ow
No
-s
nim
vi
i
ru
+H
No
ig
n-s
vi
im
ru
i
+L
ow
-s
im
i
The Number of Isolates
Figure 2.2.24. The Frequency of distribution of Burkholderia species isolated from
rice plants (Oryza sativa L. cv. Cypress) showing BPB symptoms.
Figure 2.2.25. Functional classification of bacterial isolates from rice plants
showing BPB symptoms and identified as B. glumae.
Based on the similarity/pathogenicity groupings, it looks like B. glumae
(Figure 2.2.25) and B. gladioli (Table 2.2.26) may have races based on the
distribution of pathogenicity and closeness of relatedness.
55
The Number of Isolates
25
20
15
10
5
0
i
i
i
i
i
i
im
im
im
im
im
im
s
s
s
s
s
s
h
hw
w
ig
ow
ig
ig
H
Lo
Lo
H
H
+
+
+L
+
+
u
+
u
r
u
u
u
i
u
r
r
r
i
r
ir
vi
-v
vi
vi
-v
-v
hnw
hon
w
g
o
o
i
g
N
i
N
L
Lo
H
H
Figure 2.2.26. Functional classification of the bacterial isolates from rice plants
showing BPB symptoms and identified as B. gladioli.
2.3 Summary and Conclusions
Several genera and species of bacteria were isolated from Louisiana-grown rice
showing symptoms of panicle blighting. Thirty nine bacterial species in 10 genera could
grow on the semi-specific medium, SP-G, suggesting that this medium was not unique
for selecting B. glumae and thus the species growing on the medium partly represented
the general distribution of bacteria in diseased rice tissues. Among the Burkholderia
isolates B. gladioli and B. glumae had the largest numbers of isolates highly virulent to
Cypress rice. This suggested that they were the species in the Burkholderia complex most
important in causing BPB when compared to B. multivorans, B. plantarii, and other
isolated Burkholderia and pathogenic Pseudomonas species.
The components of populations and geographic distributions of bacterial
pathogens are determined by many factors, including weather conditions, cultivar and
lines, root location, and soil types. The cultivar Cypress was previously reported to be
susceptible to B. glumae (Shahjahan et al., 2000b). Whether the cultivar was equally
susceptible to pathogenic Pseudomonas spp. needs further study. Soil type is an
important factor affecting not only the components of populations of plant-associated
bacteria, but also the variability of genetics in bacteria. It has been shown that the B.
cepacia complex displays a wide variability in genetics. Dalmastri et al. (1999)studied
the associations between the genetic diversity of maize root-associated B. cepacia
56
populations with soil type and the cultivars grown. They confirmed that soil type played a
more important role in determining the genetic diversity of B. cepacia populations than
did the cultivars grown. B. multivorans, B. gladioli, and B. glumae were found to be the
most common isolates from panicle blighted rice plants in Louisiana in this study. The
effect of soil types, cultivars, weather, and other elements affecting population dynamics
of the complex needs further study.
The fact that many non-pathogenic Psudomonas species were isolated from
diseased tissue needs further exploration, including an evaluation of the roles of
Pseudomonas species in the process of development of the syndrome and ecological
relationships with the pathogenic bacteria.
B. glumae, B. gladioli, B. multivorans, and B. plantarii strains, isolated from
diseased rice in the southern United States, caused flag leaf sheath lesions, spikelet
sterility, and poor emergence of panicles after inoculation. In most cases, they induced
discoloration and rot of leaf sheaths on rice seedlings, consistent with the literature.
Similar symptoms were reported for P. fuscovaginae causing brown-black necrosis on
stems and poor emergence of panicles (Rott et al., 1989), suggesting that some functional
characteristics, including pathogenicity, are similar to each other even though they belong
to different genera and tend to have different geographic distributions.
P. syringae pv. aptata was confirmed to attack developing florets of rice plants,
producing similar early symptoms to B. glumae and P. fuscovaginae (Goto et al., 1987).
Two pathovars of P. syringae were found in this investigation. P. syringae pv. zizanize
was pathogenic and produced symptoms similar to those produced by Burkholderia spp.
on both seedlings and panicles of rice.
Variability of bacteria for pathogenicity is a universal phenomenon, with several
possible causes including heterogeneity within strains, biochemical variability, variance
of pathogens, and loss of disease agents during inoculation. This study showed that
continued subculturing of isolates could cause loss of virulence of B. glumae. In this
survey, there were 26 Burkholderia isolates expressing non-pathogenicity on seedlings,
but pathogenicity on panicles inoculated at the late boot stage. The reverse only occurred
once. It may be that weakly virulent strains can infect the more susceptible immature
panicle tissues, but not the rapidly developing seedling tissues. As the BPB disease is
57
seedborne, avoiding contaminated seeds was essential for inoculation studies. No
infection of non-inoculated rice at the seedling or panicle stages was observed in this
study. Therefore, in this study the results of pathogenicity tests were mainly based on the
experiments on seedlings in order to ensure the reliability of results.
The BPB syndrome is poorly defined. The development of symptoms and severity
of disease not only depends on virulence of the strain, but also on environmental factors,
particularly weather conditions. Klement (1955) pointed out that P. fuscovaginae and P.
syringae grow well in relative cool, wet weather. The frequency in Japan of the
occurrence of the seedling rot disease caused by B. glumae, where the weather is cooler
and drier than Louisiana, was due to the introduction of new planting methods where
seedlings were grown in seedling boxes in warehouses maintained at high temperatures
and humidity (Goto et al., 1987).
Our research showed that a temperature range of 37oC - 41oC was a critical
environmental factor affecting the occurrence and development of the disease in the
greenhouse, not only for B. glumae, but also for B. gladioli, B. multivorans, and B.
plantarii. Therefore, in inoculation tests the descriptions of typical symptoms and the
determinations of disease severity only had significance when these environmental
conditions were met.
In this study, all of the identified isolates obtained an acceptable similarity index,
over 0.5 as assigned by the BiologTM System, and the identities could therefore be
reported according to “GN2 MicroPlateTM Instructions for Use” (Biolog Co., Hayward,
CA ). The highest indexes were received by some B. gladioli strains, which reached up to
1.0, indicating a full match with a reference strain in the database library. The 4.20
version of BiologTM database used in this study has been greatly improved as more strains
were added to the library, offering a powerful technique to support identification of
bacteria. Some Pseudomonas species were identified to the pathovar level. However, the
identification of bacteria is a long process and cross-referencing among two or more
detection methods is desirable to ensure that an isolate can be identified accurately. Fatty
acid analysis for the isolated reported on in this research is being made separately by our
laboratory.
58
Furthermore, less than 0.5 similarity indexes may not necessarily mean low
detection accuracy. It should be considered that some atypical strains have not been
collected into the database. It should also be considered that natural variability among
strains of the same bacterial species may give differences in metabolic types. There also
may be loses of some biochemical capabilities during continuing culturing and storage of
strains.
An effective control measure for plant diseases is to eradicate sources of
contamination with the pathogen. The BPB pathogens are seedborne in rice seeds
harvested the previous year (Shahjahan et al., 1998b). B. glumae could not be detected
from seeds kept outdoors for 5 months ((Tsusima et al., 1989), suggesting that low
temperature treatment may kill the bacterium. Dry heat treatment using 65oC for 6 days
or salt water selection could not ensure pathogen-free seeds in their studies. Monitoring
the contamination of commercial seed-lots seems to be more practical than chemical
control or using cold or heat treatments at this time based on current seed production,
storage, and planting practices in Louisiana. A seed treatment pesticide to control BPB is
not presently labeled for use in the United States.
Two hundred and sixty two bacterial isolates from diseased rice tissues showing
BPB symptoms were identified as Burkholderia species using the BiologTM GN2 test,
comprising 75% of the identified bacteria. Six Burkholderia species were identified
including B. glumae, B. gladioli, B. multivorans (B. cepacia genomovar II), B. plantarii,
B. vietnamiensis (B. cepacia genomovar V) and B. cocovenenans. Of them, B. gladioli,
B. multivorans, B. glumae, and B. plantarii comprised about 90 % of the Burkholderia
isolates. Pathogenicity tests on seedlings and emerging panicles showed that six species
were rice pathogens causing sheath rot or/and panicle blight. Eighty three percent of the
B. gladioli, 91% of B. glumae, 73% of B. multivorans, and 80% of B. plantarii were
pathogenic strains. It is therefore suggested that a complex of Burkholderia species
caused the BPB syndrome recently found in Louisiana. These species caused seeding
blight, seedling leaf sheath rot, and flag leaf sheath brown-black rot, sterile florets, grain
discoloration, and/or poor emergence of panicles, similar to the symptoms reported in the
literature. Symptom expression alone was not sufficient to distinguish Burkholderia
pathogens to species level.
59
This appears to be the first report of B. multivorans as a pathogen on rice.
Pathogenicity tests and data analysis showed that B. gladioli and B. glumae had more
highly virulent strains than B. multivorans and B. plantarii, suggesting that they played
the critical role in pathogenicity on rice plants.
Morphology studies showed that convex colonies with smooth margins and
yellow or gray-white in color on KB medium were common morphological
characteristics among these species. Universally, avirulent strains were associated with
gray-white colonies, supporting reports in the literature.
Pseudomonas species were shown to be one of the causes of the syndrome. They
included two P. syringae pv. zizanize, three P. fluorescens, three P. pyrrocinia, one P.
spinosa and seven P. tolaasii strains, suggesting that Pseudomonas species could also be
involved in the complex of bacterial species causing BPB on rice in Louisiana. The
factors determining whether a given bacterium is the main casual agent may include
weather conditions or cultivar and line susceptibility.
60
LITERATURE CITED
Atkins, J.G. 1974. Rice Diseases of the Americas: A Review of the Literature. pp. 55-58.
Agr. Handbook No. 448, ARS, USDA, Washington, D.C. 106p.
Ashizawa, T., S. Tsushima, H. Nemoto, N. Hayashi and H. Naito. 1997. Effect of
cleaning brown rice infected by Pseudomonas glumae. Proceedings of the Kanto-Tosan
Plant Protection Society. 44: 23-24.
Ashura, L. K., R. B. Mabagala, C. N. Mortensen. 1999. Isolation and characterization of
seed-borne pathogenic bacteria from rice (Oryza sativa L.) in Tanzania. Tanzanian
Journal of Agricultural Sciences. 2 (1): 71-80
Azegami, K., K. Nishiyama, Y. Watanabe, T. Suzuki, M. Yoshida, K. Nose and S. Toda.
1985. Tropolone as a root growth-inhibitor produced by a plant pathogenic Pseudomonas
sp. Causing seedling blight of rice. Ann. Phyto. Soc. Japan. 51: 315-317.
Azegami, K., K. Nishiyama, Y. Watanabe, I. Kadota, A. Ohuchi, and C. Fukazame. 1987.
Pseudomonas plantarii sp. Now., the causal agent of rice seedling blight. Int. J. Syst.
Bacteriol. 17: 144-152.
Azegami, K., K. Nishiyama, and H. Tabei. 1988a. Infection courts of rice seedlings with
Pseudomonas plantarii and Pseudomonas glumae. Annals of the Phytopathological
Society of Japan. 54 (3): 337-341
Azegami, K., H. Tabei, and T. Fukuda. 1988b. Entrance into rice grains of Pseudomonas
plantarii, the causal agent of seedling blight of rice. Annals of the Phytopathological
Society of Japan. 54 (5): 633-636
Azegami, K. 1996. Media and temperatures favorable for the light production by
Burkholderia glumae and B. plantarii transformed with the bioluminescence genes of
Vibrio fischeri. Annals of the Phytopathologicl Society of Japan, vol. 62 (2): 161-166
Balandreau, J., Viallard. V, B. Cournoyer, T. Coenye, S. Laevens, and P. Vandamme.
2001. Burkholderia cepacia genomovar III is a common plant-associated bacterium.
Appl. Environ. Microbiol. 67: 982-985
Baldani, V.L. D., J. I. Baldni, and J. Dobereiner. 2000. Inoculation of rice plants with the
endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. Biology and
Fertility of Soils 30 (5/6): 485-491.
61
Ball, S. F. L., and J. C. Reeves. 1991. The application of new techniques in the rapid
testing for seed-borne pathogens. Plant Varieties and Seeds 4: 169-176.
Batoko, H. 1994. Inhibition of rice (Oryza-satival) seedling elongation by a
Pseudomonas-fuscovaginae toxin. Euphytica 76 (1-2): 139-143.
Batoko, H. J. Bouharmont, and H. Maraite. 1994. Inhibition of rice (Oryza-sativa L.)
seedling elongation by a Pseudomonas fuscovaginae toxin. Euphytica 76(1-2): 139-143.
Batoko, H. 1997. Involvement of toxins produced by Pseudomonas fuscovaginae in
aetiology of rice bacterial sheath brown rot. Journal of PhytopatologyPhytopathologische Zeitschrift. 145(11-12): 525-531.
Bauernfeind, A., I. Schneider, R. Jungwirth, and C. Roller. 1998. Discrimination of
Burkholderia gladioli from other Burkholderia species detectable in cystic fibrosis
patients by PCR. Journ. of Clinical Microbiology. 36 (9): 2748-2751
Baxter, I. A., P. I. Lamber and I. N. Simpson. 1997. Isolation from clinical sources of
Burkholderia cepacia possessing characteristics of Burkholderia gladioli. Journ. of
Antimicrobial Chemotherapy. 39: 169-175
Bevivino, A., S. Sarrocco, C. Dalmastri, S. Tabacchioni, C. Cantale, and L. Chiarini.
1998. Characterization of a free-living maize-rhizosphere population of Burkholderia
cepacia: effect of seed treatment on disease suppression and growth promotion of maize.
FEMS Microbiol. Ecol. 27:225-237
Brock, T. D., Madigan, M. T., Martinko, J. M., and J. Parker. 1994. Biology of
Microbiology. 7th edition. Prentice-Hall, Inc. A Paramount Communications Company
Englewood Cliffs, New Jersey 07632. pp. 45-46.
Buckle, K. A. and E. Kartadarma. 1990. Inhibition of bongkrek acid and toxoflavin
production in tempe bongkrek containing Pseudomonas cocovenenans. Journal of
Applied Bacteriology 68: 571-576.
Burkholder, W. H. 1949. Sour skin, a bacterial rot of onion bulbs. Phytopathology 32:
141-149.
Campbell, P. W., J. A. Phillips, G. J. Heidecker, M. R. S. Krishnamani, R. Zahorchak,
and T. L. Stull. 1995. Detection of Pseudomonas (Burkholderia) cepacia using PCR.
Pediatr. Pulmonol. 20: 44-49
62
Candlish, A. A. G., J. D. Taylor, and J. Cameron. 1988. Immunological methods as
applied to bacterial pea blight. Brighton Crop Protection Conference- Pests and Diseases
(1988). 2: 787-794.
Chen, Z. and T. W. Mew. 1998. Relationship between the colonization, concentration and
spray timing of antagonistic bacteria and sheath blight of rice. Jiangsu Journ. of Agr. Sci.
14 (1): 31-35.
Cheng, G., A. K. M. Shahjahan, X. L. Yuan, and M. C. Rush. 2001. Distinguishing
among isolates of Burkholderia glumae, B. gladioli, B. plantarii, and B. cepacia from
Rice. Proc. 29th RTWG. 29: 94.
Chiarini, L., A. Bevivino, S. Tabacchioni, and C. Dalmastri. 1998. Inoculation of
Burkholderia cepacia, Pseudomonas fluorescens and Enterobacter sp. on Sorghum
bicolor: root colonization and plant growth promotion of dual strain inocula. Soil Biol.
Biochem. 30: 81-87
Chien, C. C., and Y. C. Chang. 1987. The susceptibility of Rice Plants at different growth
stages and of 21 commercial rice varieties to Pseudomonas glumae. Journ. of Agric. Res.
of China 36 (3): 302-310
Coenye, T., P. Vandamme, J. R. W. Govan, and J. J. LiPuma. 2001. Taxonomy and
identification of the Burkholderia cepacia complex. Journ. of Clinical Micro. 39: 34273436.
Cottyn, B., M. T. Cerez, M. F. Van Outryve, J. Barroga, J. Swings, and T. W. Mew.
1996a. Bacterial diseases of rice. I. Pathogenic bacteria associated with sheath rot
complex and grain discoloration of rice in the Philippines. Plant Dis. 80 (4): 429-437
Cottyn, B., M. F. van. Outryve, M. T. Cerez, M. de. Cleene, J. Swings, and T. W. Mew.
1996b. Bacterial disease of rice. II. Characterization of pathogenic bacteria associated
with sheath rot complex and grain discoloration of rice in the Philippines. Plant Dis. 80
(4): 438-445.
Cottyn, B., E. Regalado, B. Lanoot, M. de. Cleene, T. W. Mew, and J. Swings. 2001.
Bacterial populations associated with rice seed in the tropical environment.
Phytopathology 91 (3): 282-292.
Dalmastri, C., L. Chiarini, C. Cantale, A. Bevivino, and S. Tabacchioni. 1999. Soil type
and maize cultivar affect the genetic diversity of maize root-associated Burkholderia
cepacia populations. Microbial Ecology 38: 273-284.
63
Daubaras, D. L., C. E. Danganan, A. Hübner, R. W. Ye, W. Hendrickson, and A. M.
Chakrabarty. 1996. Biodegradation of 2, 4, 5-trichlorophenoxyacetic acid by
Burkholderia cepacia strain AC1100: evolutionary insight. Gene 179:1-8
Duveiller, E., K. Miyajima, F. Snacken, A. Autrique, and H. Maraite. 1988.
Characterization of Pseudomonas fuscovaginae and differentiation from other fluorescent
pseudomonas occurring on rice in Burundi. J. Phytopathology 122: 97-107.
Estrada-de los santos, P., R. Bustillos-cristales, and J. Caballero-mellado. 2001.
Burkholderia, a genus rich in plant-associated nitrogen fixers with wide environmental
and geographic distribution. Applied and Environ. Micro. 67: 2790-2798.
Flamand, M. C. 1996. Production of syringotoxin and other bioactive peptides by
Pseudomonas fuscovaginae. Physiol. and Mole. Plant Path. 48 (4): 217-231.
Furuya, N., T. Okamoto, Y. Kori, N. Matsuyama, and S. Wakimoto. 1991. Control of
bacterial seedling rot of rice by avirulent strains of Pseudomonas glumae. Ann. of the
Phytopath. Soc. of Jap. 57 (3): 371-376
Furuya, N., H. Matsuo, K. Koga, N. Matsuyama. 1992. Control of bacterial seedling rot
of rice by pretreatment with avirulent strains of Pseudomonas glumae and its
mechanisms. Proc. of the Kanto-Tosan Plant Prot. Soc. 38: 4-6.
Furuya, N. 1992. Production of antibacterial substances by Pseudomonas-glumae. Journ.
of the Fac. of Agric. Kyushu Univ. 37 (2): 149-158
Furuya, N., K. Liyama, Y. Ueda, and N. Matsuyama. 1997. Reaction of tobacco and rice
leaf tissue infiltrated with Burkholderia glumae or B. gladioli. Journ. of the Fac. of Agric.
Kyushu Univ. 42 (1-2): 43-51.
Furuya, N., H. Ura, K. Ilyama, M. Matsumoto, M. Takeshita and Y. Takanami. 2002.
Specific oligonucleotide primers based on sequences of the 16S-23S rDNA spacer region
for the detection of Burkholderia gladioli by PCR. J. Gen. Plant Pathol. 68: 220-224.
Gillis, M., T. V. Van, R. Bardin, M. Goor, P. Hebra, A. Willems, P. Segers, K. Kersters,
T. Heulin, and M. P. Fernandez. 1995. Polyphasic taxonomy in the genus Burkholderia
leading to an emended description of the genus and proposition of Burkholderia
vietnamiensis sp. nov. for N2-fixing isolates from rice in Vietnam. Int. Journ. of
Systematic Bact. 45 (2): 274-289.
64
Goto, K., and K. Ohata. 1956. New bacterial disease of rice (brown stripe and grain rot).
Ann. Phyto. Soc. Jap. 21: 46-47.
Goto, T., K. Nishiyama and K-I. Ohata. 1987. Bacteria causing grain rot of rice. Ann.
Phytopath. Soc. Jap. 53: 141-149.
Groth, D. E. and D. M. Brandon. 1984. Effect of N. fertilizer on disease development in
rice. In. Ann. Prog. Rep., Rice Exp. Stn., Crowley, LA 76: 233-243.
Groth, D. E. and C. A. Hollier. 1984. Rice disease survey. In. Ann. Prog. Reg., Rice Exp.
Stn., Crowley, LA 77: 250-257.
Groth, D.E., M.C. Rush, and C.A. Hollier. 1991. Rice Diseases and Disorders in
Louisiana. Bulletin No. 828, Louisiana State University Agricultural Center. Baton
Rouge, Louisiana. 37 p.
Hashioka,Y. 1969. Rice diseases in the world – III Bacterial diseases. Il Riso18: 189-204.
Hanzawa, S. and K. Kaji. 1991. Secondary infection of rice seedling rot in a nursery box,
caused by grain rot bacterium, Pseudomonas glumae Kurita and Tabei 1967 and rot of
diseased seedlings in paddy field. Ann. Rept. Plant Prot. North Jap. 42: 27-30.
Hebbar, K. P., M. H. Martel, and T. Heulin. 1998. Suppression of pre- and
postemergence damping-off in corn by Burkholderia cepacia. Eur. J. Plant Pathol. 104:
29-36
Hikichi, Y., C. Noda, and K. Shimizu. 1989. Oxolinic acid. Jap. Pest. Info. 55: 21-23.
Hikichi, Y. 1993a. Mode of action of oxolinic acid against bacterial seedling rot of rice
caused by Pseudomonas glumae. I. Relationship between population dynamics of P.
glumae on seedling of rice and disease severity of bacterial seedling rots of rice. Ann.
Phyto. Soc. Jap. 59: 441-446.
Hikichi, Y. 1993b. Relationship between population dynamics of Pseudomonas glumae
on rice plants and disease severity of bacterial grain rot of rice. J. Pesticide Sci.18: 319324.
Hikichi, Y. 1993c. Antibacterial activity of oxolinic acid on Pseudomonas glumae.
Annals of the Phytopath. Soc. of Jap. 59 (4): 369-374.
65
Hikichi, Y., T. Okuno, and I. Furusawa. 1993. Immunofluorescent antibody technique for
detecting Pseudomonas glumae on rice plants. Annals of the Phytopath. Soc. of Jap. 59
(4): 477-480
Hikichi, Y., T. Okuno, and I. Furusawa. 1994. Susceptibility of rice spikelets to infection
with Pseudomonas glumae and its population-dynamics. Journ. of Pest. Sci. 19 (1): 1117.
Hikichi, Y. 1995a. Mode-of-action of oxolinic acid against bacterial seedling rot of rice
caused by Pseudomonas glumae. II. Efficacy of oxolinic acid against secondary infection.
Annals of the Phytopath. Soc. of Jap. 59 (4): 447-451
Hikichi, Y. 1995b. The spread of bacterial grainrot of rice and its control in the paddy
fields. J. Pest. Sci. 20: 329-331.
Hikichi, Y. and H. Egami. 1995. Control systems for bacterial grain rot of rice with
oxolinic acid and seed selection with salt solution. Annals of the Phytopath. Soc. of Jap.
61 (4): 405-409
Hikichi, Y., T. Okuno, and I. Furusawa. 1995a. Mode of action of oxolinic acid against
bacterial seedling rot of rice caused by Pseudomonas glumae. I: Infection with P. glumae
into plumules. Annals of the Phytopath. Soc. of Jap. 61 (2): 134-136
Hikichi, Y., T. Okuno and I. Furusawa. 1995b. Mode of action of oxolinic acid against
bacterial seedling rot of rice caused by Pseudomonas glumae. III. Infection with P.
glumae into plumules. Ann. Phyto. Soc. Japan. 61: 134-136.
Hikichi, Y., H. Egami, Y. Oguri, and T. Okuno. 1998. Fitness for survival of
Burkholderia glumae resistant to oxolinic acid in rice plants. Ann. Phyto. Soc. Jap. 64
(3): 147-152.
Holmes, A., J. Govan, and R. Goldstein. 1998. Agricultural use of Burkholderia
(Pseudomonas) cepacia: a threat to human health? Emerg. Infect. Dis. 4: 221-227
Hu, F. P., J. M. Young, and C. M. Triggs. 1991. Numerical analysis and determinative
tests for nonfluorescent plant-pathogenic Pseudomonas spp. and genomic analsysis and
reclassification of species related to Pseudomonas avenae manns 1909. Int. Journ. of
Systematic Bact. 41: 516-525.
66
Iguchi, K. 1991. Growth development of rice plant suffering from bacterial seedling
blight after transplanting and the population changes of the causal agent, Pseudomonas
plantarii in rice plant. Proceed. of the Kanto-Tosan Plant Protect. Soc. 38: 23-24.
Iguchi, K. 1992. SEM observation of the infection site on rice seedling infected with
Pseudomonas plantarii. Proceed. of the Kanto-Tosan Plant Protec. Soc. 39: 17-19.
Iiyama, K., N. furuya, Y. Takanami and N. Matsuyama. 1995. A role of phytotoxin in
virulence of Pseudomonas glumae Kurita et Tabei. Ann. Phyto. Soc. Japan. 61: 470-476.
Iiyama, K., N. Furuya, H. Ura, and N. Matsuyama. 1998. Role of phytotoxin in the
pathogenesis of Burkholderia species. Journ. of the Fac. Agric. Kyushu Univ. 42 (3/4):
289-293.
Isogawa, Y., S. Kato, and S-H. Li. 1989. Studies on environmental factors and control
methods of bacterial grain rot of rice caused by Pseudomonas glumae Kurita te Tabei II.
Res. Bull. Aichi. Agric. Res. Ctr. 21: 85-90.
Iwai, T., H. Kaku, R. Honkura, S. Nakamura, H. Ochiai, T. Sasaki, and Y. Ohashi. 2002.
Enhanced resistance to seed-transmitted bacterial diseases in transgenic rice plants
overproducing an oat cell-wall-bound thionin. Mole. Plant Microbe Interaction 15: 515521
Jaunet, T. 1996. Pathogenicity of process of Pseudomonas fuscovaginae, the causal agent
of sheath brown rot of rice. Journ. of Phytopathology-Phytopathologische Zeitschrift 144
(9-10): 425-430
Jeong, Y., J. Kim, S. Kim, Y. Kang, T. Nagamatsu, and I. Hwang. 2003. Toxoflavin
produced by Burkholderia glumae causing rice grain rot is responsible for inducing
bacterial wilt in many field crops. Plant Dis. 87:890-895.
Jones, J. B. 1993. Evaluation of the Biolog GN MicroPlate system for identification of
some plant-pathogenic bacteria. Plant Dis. 77(6): 553-558
Jurita, T. and H. Tabei. 1967. On the pathogenic bacterium of bacterial grain rot of rice.
Ann. Phyto. Soc. Jap. 33: 111.
Kadota, I. And A. Ohuchi. 1983. Symptoms of bacterial brown stripe of rice seedlings in
nursery boxes. Ann. Phyto. Soc. Jap. 49: 561-564.
67
Kadota, I. 1996. Studies on the pathogen of bacterial brown stripe of rice and its ecology.
Bull. of the Hokuriku Nat. Agr. Expt. Sta. 38: 113-171
Kadota, I., A. Mizuno and K. Nishiyama. 1996. Detection of a protein specific to the
strain Pseudomonas avenae Manns 1909 pathogenic to rice. Ann. Phyto. Soc. Jap. 62:
425-428.
Karpati, F., and J. Jonasson. 1996. Polymerase chain reaction for the detection of
Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia in
sputum of patients with cystic fibrosis. Mol. Cell. Probes 10:397-403
Katoh, T., Y. Fujita and H. Sakuma. 1990. Tropolone method to find rice seed infected
with bacterial seedling blight, Pseudomonas plantarii. Ann. Rept. Plant Prot. North Jap.
41: 47-49.
Katsube, K. and S. Takeda. 1997. Suppression of bacterial seedling rot and bacterial
seedling blight of rice on the pool nursery. Ann. Rept. of the Soc. of Plant Protect. of
North Jap. 48: 43-46
Katsube, K., and S. I. Takeda. 1998. Effect of copper content in seed disinfectants on
control efficacy against bacterial seedling rot [Burkholderia glumae] and bacterial
seedling blight [B. plantarii] of rice. Ann. Repo. Soc. Plant Prot. North Jap. 49: 33-36.
Katsube, K. and S. Takeda. 1998. Increase in severity of bacterial seedling blight of rice
with soil drenching of fungicides. Ann. Rept. Plant Prot. North Jap. 49: 37-39.
Kawaradani, M., K. Okada and S. Kusakari. 2000. New selective medium for isolation of
Burkholderia glumae from rice seeds. J. Gen. Plant Pathol. 66: 234-237.
Klement, A. 1955. A new bacterial disease of rice caused by Pseudomonas oryzicola n.
sp. Acta Microbiol. Acad. Sci. Hung. 2: 265-274.
Klement, Z. 1983. Detection of seed-borne bacteria by hypersensitive reaction. Seed Sci.
and Tech. 11: 589-593
Kodama, K., N. Kimura, and K. Komagata. 1985. Two new species of Pseudomonas: P.
oryzihabitans isolated from rice paddy and clinical specimens and P. luteola isolated
from clinical specimens. Int. Journ. of Systematic Bact. 35 (4): 467-474.
Kurita, T., H. Tabei, and T. Sato. 1964. A few studies on factors associated with infection
of bacterial grain rot of rice. Ann. Phyto. Soc. Jap. 29: 60
68
Kurita, T. and H.Tabei. 1967. On the pathogenic bacterium of bacterial grain rot of rice.
Ann. Phyto. Soc. Jap. 33: 111.
Kuwata, H. 1985. Pseudomonas syringae pv. oryzae pv. Nov., causal agent of bacterial
halo blight of rice. Ann. Phytopath. Soc. Jap. 51: 212-218.
Lee, F.N. 1992a. Ear blight. p.32. In: Compendium of Rice Diseases. (Ed.) Webster, R.K.
and P.S. Gunnell. APS Press. St. Paul, MN. 62p.
Lee, F.N. 1992b. Grain discoloration. pp.31-32. In: Compendium of Rice Diseases. (Ed.)
Webster, R.K. and P.S. Gunnell. APS Press. St. Paul, MN. 62p.
Lindberg, G. D., J. M. Larkin and H. A. Whaley. 1980. Production of tropolone by a
Pseudomonas. J. Nat. Prod. 43: 592-594.
Lindberg, G. D. 1981. An antibiotic lethal to fungi. Plant Dis. 65: 680-683.
LiPuma, J. 1998. International Burkholderia cepacia Working group. Available: onlinehttp://allserv.rug.ac.be/~tcoenye/cepacia/miami.pdf
Liyama, K., N. Furuya, K. Hara, N. Nakashima, Y. Takanami, and N. Matsuyama. 1994.
Phytotoxin produced by Pseudomonas glumae Jurita et Tabei, a causal bacterium of the
grain and seedling rot of rice. Journ. of the Fac. of Agr., Kyushu Univ. 38 (3-4): 175-181.
Liyama, K., N. Furuya, Y. Takanami, and N. Matsuyama. A role of phytotoxin in
virulence of Pseudomonas glumae Jurita et Tabei. 1995. Annals of the Phytol. Soc. of
Jap. 61 (5): 470-476.
Liyama, K., N. Furuya, H. Ura, and N. Matsuyama. 1998. Role of phytotoxins in the
pathogensis of Burkholderia species. Lab. of Plant Path., Fac. of Agr., Kyushu Univ. 42
(3/4): 289-293.
LSU Agricultural Center. 1987. Pub. 2321, Rice Production Handbook. p.39. Louisiana
Agric. Expt. Sta. Baton Rouge, LA. 63p.
LSU Agricultural Center. 1999. Louisiana Rice Production Handbook. p.81. La. State
Univ. Agr. Center. Baton Rouge, LA. 116p.
69
Lu, J.J. and T.T. Chang. 1980. Rice in its temporal and spatial perspectives. In: Rice:
Production and Utilization. (Ed.) B.S. Luh. AVI Publishing Co., Inc. Westport, CN.
925p.
Matsuda, I. And Z. Sato. 1987. Ecology of P. glumae, cause bacterial grain rot of rice,
from planting to the mature stage. Ann. Phyto. Soc. Jap. 53:122.
McLoughlin, T. J., J. P. Quinn, A. Bettermann, and R. Booklands. 1992. Pseudomonas
cepacia suppression of sunflower wilt fungus and role of antifungal compounds in
controlling the disease. Appl. Environ. Microbiol. 58: 1760-1763.
Mew, T. W. 1992. Grain Rot. p.9. In: Compendium of Rice Diseases. (Ed.) Webster,
R.K. and P.S. Gunnell. APS Press, St. Paul, MN. 62p.
Miyagawa, H. and S. Takaya. 2000. Biological control of bacterial grain rot of rice by
avirulent strain of Burkholderia gladioli. Bull. of the Chugoku Nat. Agr. Expt. Sta. 21: 121.
Miyagawa, H. 2000. Biocontrol of bacterial seedling blight of rice caused by
Burkholderia gladioli using with its avirulent isolate. Ann. Phyto. Soc. Jap. 66: 232-238.
Miyagawa. H. 2000. Specific detection of pigment-producing strains of Burkholderia
glumae by ammonia treatment. Ann. Phyto. Soc. Jap. 66: 246-251.
Miyajima, K., A. Tanii, and T. Akita. 1983. Pseduomonas fuscovaginae sp. Nov., nom.
Rev. Int. J. Syst. Bacteriol. 33: 656-657.
Mizuno, A., S. Tsushima, I. Kadota and K. Nishiyama. 1995. A cloned DNA probe for
detection of Pseudomonas gladioli. Ann. Phyto. Soc. Jap. 61: 22-26.
O'Callaghan, E. M., M. S. Tanner, and G. J. Boulnois. 1994. Development of a PCR
probe test for identifying Pseudomonas aeruginosa and Pseudomonas (Burkholderia)
cepacia. J. Clin. Pathol. 47: 222-226.
Ohno, Y., S. Okuda, T. Natsuaki, and M. Teranaka. 1992. Control of bacterial seedling
blight of rice by fluorescent Pseudomonas spp. Proceedings of the Kanto-Tosan Plant
Protect. Soc. 39: 9-11.
Ohta, K. and R. Tei. 1994. Chemical control of bacterial seedling blight and bacterial
grain rot of rice in nursery stage. Proceed. of the Kanto-Tosan Plant Protect. Soc. 41: 911.
70
Ohta, K. 1995. A simple detection method for seed infected with bacterial seedling blight
pathogen. Proceed. of the Kanto-Tosan Plant Protect. Soc. 42: 15-17.
Ohta, k. 1995. Control of bacterial seedling blight of rice by vacuum soaking method.
Proceed. of the Kanto-Tosan Plant Protect. Soc. 42: 19-21.
Otofuji, M., K. Kadoshige and K. Yoshida. 1988. Persistent part of Pseudomonas glumae
Jurita et Tabei in rice plant. Proc. Assoc. Plant Prot. Kyushu. 34: 1-4.
Ou, S. M. 1985. Rice Disease. 2nd. Ed. Commonwealth Mycological Institute, Kew,
Surrey, England. 380p.
Palleroni, N. J. and B. Holmes. 1981. Pseudomonas cepacia sp. nov., nom. rev. Int.
Journ. of Systematic Bact.. 31 (4): 479-481.
Parke, J. L. and D. Gurian-Sherman. 2001. Diversity of the Burkholderia cepacia
complex and implications for risk assessment of biological control strains. Annu. Rev.
Phytopathol. 39: 225-258.
Parke, J. L. 1998. Burkholderia cepacia: Friend or Foe? Available: Online:
http://www.apsnet.org/online/feature/.
Pelt, C. V., C. M. Verduin, V. H. F. Goessens, M. C. Vos, B. Tummler, C. Segonds, F.
Reubsaet, H. Verbrugh, and A. V. Belkum. 1999. Identification of Burkholderia spp. in
the clinical microbiology laboratory. Comparison of Conventional and Molecular
Methods. 37: 2158-2164.
Reiter, B., A. Glieder, D. Talker, and H. Schwab. 2000. Cloning and characterization of
EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes. Appl.
Microbiol. Biotechnol. 54: 778-785.
Rott, P., J. L. Notteghem, and P. Frossard. 1989. Identification and characterization of
Pseudomonas fuscovaginae, the causal agent of bacterial sheath brown rot of rice, from
Madagascar and other countries. Plant Dis. 73: 133-137.
Rott, P., J. Honegger, J.-L. Notteghem, and S. Ranomenjanahary. 1991. Identification of
pseudomonas-fuscovaginae with biochemical, serological, and pathogenicity tests. Plant
Dis. 75 (8): 843-846.
Rush, M. C., C. Clark, and D. E. Groth. 1998. Rice bacterial leaf blight found in
Louisiana rice. Proc. 22nd RTWG. 22: 65.
71
SAS. 1982. SAS Users Guide: Statistics. Statistical Analysis System, Car, North
Carolina. 584 pp.
Sato, Z., Y. Koiso, S. Iwasaki, I. Matsuda, and A. Shirata. 1989. Toxins produced by
Pseudomonas glumae. Ann. Phyto. Soc. Jap. 55: 353-356.
Segonds, C., T. Heulin, N. Marty, and G. Chabanon. 1999. Differentiation of
Burkholderia species by PCR-restriction fragment length polymorphism analysis of the
16S rRNA gene and application to cystic fibrosis isolates. J. Clin. Microbiol. 37: 22012208
Seki, M. 1959. Study on the bacterial grain rot of rice. Bull. Saga Pref. Agric. Exp. Stn. 2:
131-147.
Shahjahan, A. K. M., M. C. Rush, C. E. Clark, and D. E. Groth. 1998. Bacterial sheath rot
and panicle blight of rice in Louisiana. Proc. 27th RTWG. 27: 31-32.
Shahjahan, A. K. M., D. E. Groth, C. Clark, S. D. Linscombe, and M. C. Rush. 2000a.
Critical stage of infection and the effect of infected seeds on disease development and
yield of rice. Proc. 28th RTWG. 28: 77.
Shahjahan, A. K., M. C. Rush, D. Groth, and C. Clark. 2000b. Panicle Blight. Rice Journ.
15: 26-29.
Shahjahan, A. K. M., D. E. Groth, C. A. Clark, S. D. Linscombe, and M. C. Rush. 2000c.
Epidemiological studies on panicle blight of rice: critical stage of infection and the effect
of infected seeds on disease development and yield of rice. Proc. 28th RTWG. 28: 77.
Shakya, D.D. and H. S. Chung. 1983. Detection of Pseudomonas avenae in rice seed.
Seed Sci. & Tech. 11: 583-587.
Shakya, D. D., F. Vinther and S. B. Mathur. 1985. World wide distribution of a bacterial
stripe pathogen of rice identified as Pseudomonas avenae. Phytopath. Z. 114: 256-259.
Shubba, K., S. A. Shetty, H. S. Shetty and N. G. K. Karanth. 1992. Interactions between
seed-borne bacteria and fungi of paddy. Int. J. Trop. Plant Dis. 10: 125-130.
Simpson, I. N., J. Finlay, D. J. Winstanley, N. Dewhurst, J. W. Nelson, S. L. Butler and J.
R. W. Govan. 1994. Multi-resistance isolates possessing characteristics of both
Burkholderia (Pseudomonas) cepacia and Burkholderia gladioli from patients with cystic
fibrosis. Journ. of Antimicrobial Chemotherapy 34: 353-361.
72
Sogou, K. and Y. Tsuzaki. 1983. Overwintering of grain rot bacterium Pseudomonas
glumae and its infection to rice plant in the paddy field. Proc. Assoc. Plant Prot. Shikotu.
18: 15-20.
Song, W. Y. and K. HyungMoo. 1999. Current status of bacterial grain rot of rice in
Korea. Res. in Plant Dis. 5 (1): 1-7.
Stead, D. E. 1992. Grouping of plant-pathogenic and some other Pseudomonas spp. by
using cellular fatty acid profiles. Int. Journ. of Systematic Bact.. 42 (2): 281-295.
Sthapit, B. 1995. Inheritance and selection of field-resistance to sheath brown-rot disease
in rice. Plant Dis. 79 (11): 1140-1144.
Suslow, T. V., M. N. Schroth, and M. Isaka. 1982. Application of a rapid method for
Gram differentiation of plant pathogenic and saprophytic bacteria without staining.
Phytopathology 72: 917-918.
Suzuki, F., Y. F. Zhu, H. Sawada, and I. Matsuda. 1998. Identification of proteins
involved in toxin production by Pseudomonas glumae. Annals of the Phytopath. Soc. of
Jap. 64 (2): 75-79
Tabacchioni, S., A. Bevivino, L. Chiarini, P. Visca, and M. Del Gallo. 1993.
Characteristics of two rhizosphere isolates of Pseudomonas cepacia and their potential
plant-growth-promoting activity. Microb. Rel. 2:161-168.
Tabei, H., K. Azegami, T. Fukuda, and T. Goto. 1989. Stomatal infection of rice grain
with Pseudomonas glumae, the causal agent of the bacterial grain rot of rice. Ann. Phyto.
Soc. Jap. 55 (2): 224-228.
Takao, G. 1987. Bacteria causing grain rot of rice. Annals of the Phytopath. Soc. of Jap.
53: 141-149.
Takeuchi, T. 1994. A method for testing the effect of seed disinfection for bacterial
seedling blight of rice. Ann. Rept. Plant Prot. North Jap. 45: 21-26.
Takeuchi, T. Influence of cultural conditions in nursery bed on occurrence of bacterial
seedling blight of rice. Ann. Rept. Plant Prot. North Jap. 45: 17-20.
Takeuchi, T., H. Sawada, F. Suzuki, and I. Matsuda. 1997. Specific detection of
Burkholderia plantarii and B. glumae by PCR using primers selected from the 16S-23S
rDNA spacer regions. Annals of the Phytopath. Soc. of Jap. 63 (6): 455-462.
73
Tanaka, T., T. Katoh, and Y. Fujita. 1994. Isolation of Pseudomonas plantarii, pathogen
of bacterial seedling blight of rice, from the weeds around the paddy field. Annals of the
Phytopath. Soc. of Jap. 60 (5): 640-643.
Tanii, A., T. Baba and T. Haruki. 1974. Bacteria isolated from black rot of rice grains.
Ann. Phytopath. Soc. Jap. 40: 309-318.
Tanii, A., K. Miyajima, and T. Akita. 1976. The sheath brown rot disease of rice plant
and its causal bacterium, Pseudomonas fuscovaginae, A. Tanii, K. Miyajima, et T. Akita
sp. Nov. Ann. Phyto. Soc. Jap. 42: 540-548.
Taxahash, C. 1993. Effect of autoclaving of nursery bed soil on occurrence of bacterial
seedling blight Pseudomonas plantarii Vzegara et al. Ann. Rept. Plant Prot. North Jap.
44: 14-15.
Tominaga, T., K. Kimura, and N. Goh. 1983. Bacterial brown stripe of rice in nursery
box, caused by Pseudomonas avenae. Ann. Phytopath. Soc. Jap. 49: 463-466.
Tran Van, V. 1994. Growth promotion of rice after inoculation with the nitrogen-fixing
bacterium Burkholderia vietnamiensis isolated from an acid-sulfate soil in Vietnam.
Agronomie 14 (10): 697-707
Tran Van, V., O. berge, S. Ngo Ke, J. Balandreau and T. Heulin. 2000. Repeated
beneficial effects of rice inoculation with a strain of Burkholderia vietnamiensis on early
and late yield components in low fertility sulphate acid soils of Vietnam. Plant and Soil
218: 273-284.
Trung, H. M., N. V. Van, N. V. Vien, D. T. Lam, and M. Lien. 1993. Occurrence of rice
grain rot disease in Vietnam. Int. Rice Res. Notes 18 (3): 30
Tsushima, S., S. Mogi and H. Saito. 1985. Effects of inoculum density, incubation
temperature and incubation period on the development of rice bacterial grain rot. Proc.
Assoc. Plant Prot. Kyushu. 31: 11-12.
Tsushima, S., S. Wakimoto and S. Mogi. 1986. Selective medium for detecting
Pseudomonas glumae Jurita et Tabei, the causal bacterium of grain rot of rice. Ann.
Phytopath. Soc. Japn. 52: 253-259.
Tsushima, S., K. Tsuno, S. Mogi, S. Wakimoto and H. Saito. 1987. The multiplication of
Pseudomonas glumae on rice grains. Ann. Phyto. Soc. Japan. 53: 663-667.
74
Tsushima, S., S. Mogi, and H. Naito, and H. Saito. 1989. Existence of Pseudomonas
glumae on the rice seeds and development of the simple method for detecting P. glumae
from the rice seeds. Bull. of the Kyushu Nat. Agric. Expt. Sta. 25 (3): 261-270.
Tsushima, S. 1991. Reduction of virulence and colonial variation of Pseudomonas
glumae cultured on PSA medium. Bull. of the Jyushu Nat. Agric. Expt. Sta. 26 (4): 361379.
Tsushima, S. and H. Naito. 1991. Spatial distribution and dissemination of bacterial grain
rot of rice caused by Pseudomonas glumae. Ann. Phyto. Soc. Jap. 57 (2): 180-187.
Tsushima, S., S. Mogi, H. Naito, and H. Saito. 1991. Populations of Pseudomonas
glumae on rice plants. Ann. Phyto. Soc. Jap. 57: 145-152.
Tsushima, S., C. Narimatsu, A. Mizuno, and R. Kimura. 1994. Cloned DNA probes for
detection of Pseudomonas glumae causing bacterial grain rot of rice. Ann. Phyto. Soc.
Jap. 60: 576-584.
Tsushima, S., H, Naito, and M. Koitabashi. 1995a. Change in panicle susceptibility
associated with flowering rate of spikelets in bacterial grain rot of rice caused by
Pseudomonas glumae. Annals of the Phytopath. Soc. of Jap. 61 (2): 109-113.
Tsushima, S., H. Naito, and M. Koitabashi. 1995b. Forecast of yield loss suffered from
bacterial grain rot of rice in paddy fields by severely diseased panicles. Annals of the
Phytopath. Soc. of Jap. 61 (5): 419-424.
Tsushima, S. 1996. Epidemiology of bacterial grain rots of rice caused by Pseudomonas
glumae. JARQ 30 (2): 85-89.
Tsushima, S., H. Naito, and M. Koitabashi. 1996. Population dynamics of Pseudomonas
glumae, the causal agent of bacterial grain rot of rice, on leaf sheaths of rice plants in
relation to disease development in the field. Ann. Phyto. Soc. Japan. 62: 108-113.
Tyler, S. D., C. A. Strathdee, K. R. Rozee, and W. M. Johnson. 1995. Oligonucleotide
primers designed to differentiate pathogenic Pseudomonads on the basis of the sequence
of genes coding for 16S-23S rRNA internal transcribed spacers. Clin. Diagn. Lab.
Immunol. 2: 448-453.
Uematsu, T., D. Yoshimura, K. Nishiyama, T. Ibaraki, and H. Fuji. 1976. Pathogenic
bacterium causing seedling rot of rice. Ann. Phyto. Soc. Jap. 42: 461-471.
75
Uematsu, T. D. Yoshimura, K. Nishiyama, T. Ibaraki, and H. Fujii. 1976. Occurrence of
bacterial seedling rot in nursery flat, caused by grain rot bacterium Pseudomonas glumae.
Ann. Phyto. Soc. Jap. 42: 310-312.
Ura, H., M. Matsumoto, K. Liyama, N. Furuya, and N. Matsuyama. 1998. PCR-RFLP
analysis for distinction of Burkholderia species, causal agents of various rice diseases.
Proceed. of the Assoc. for Plant Protect. of Kyushu Univ. 44 (11): 33-66.
Urakami, T., C. Ito-Yoshida, H. Araki, T. Kijima, K-I. Suzuki, and K. Komagata. 1994.
Transfer of Pseudomonas plantarii and Pseudomonas glumae to Burkholderia as
Burkholderia spp. and description of Burkholderia vandii sp. nov. Int. Journ. of
Systematic Bact. 44 (2): 235-245.
Valter, H. B. 1949. Sour skin, a bacterial rot of onion bulbs. Phytopathology 40: 115117.
Vandamme, P. A. R. 1996. Polyphasic taxonomy in practice: the Burkholderia cepacia
challenge. Available: online: http://www.wfcc.info/NEWSLETTER/.
Vandamme, P., B. Holmes, M. Vancanneyt, T. Coenye, B. Hoste, R. Coopman, H.
Revets, S. Lauwers, M. Gillis, K. Kersters, and J. R. W. Govan. 1997. Occurrence of
multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of
Burkholderia multivorans sp. nov. Int. J. Syst. Bacteriol. 47: 1188-1200.
Vandamme, P., E. Mahenthiralingam, B. Holmes, T. Coenye, B. Hoste, P. D. Vos, D.
Henry, and D. P. Speert. 2000. Identification and population structure of Burkholderia
stabilis sp. nov. (formerly Burkholderia cepacia genomovar IV). Journ. of Clinical
Microbiol. 38 (3): 1042-1047.
Wakimoto, S., A. Makoto, and K. Tscchiya. 1987. Serological specificity of
Pseudomonas glumae, the pathogenic bacteria of grain rot disease of rice. Annals of the
Phytopath. Soc. of Jap. 53: 150-158.
Wang, Z., R. Yanagita, K. Tsuchiya, N. Matsuyama and S. Wakimoto. 1991.
Relationship between productivity and some other bacteriological properties in the
mutant strains of Pseudomonas glumae induced by nitrosoguanidine-treatment. Ann.
Phyto. Soc. Japan. 57: 219-224.
Wasano, K. and S-I. Okuda. 1994. Evaluation of resistance of rice cultivars to bacterial
grain rot by the syringe inoculation method. Breeding Sci. 44: 1-6.
76
Watanabe, Y., S. Okuda, T. Natsuaki and M. Teranaka. 1991. Chemical control of
bacterial seedling blight of rice and the influence of cultural conditions on its occurrence.
Proceed. of the Kanto-Tosan Plant Protect. Soc. 38: 19-21.
Welch, D. F. 1991. Applications of cellular fatty acid analysis. Clinical Microb. Rev. 4:
422-438.
Weller, D. M. 2002. Microbial populations responsible for specific soil suppressivness to
plant pathogens. Annu. Rev. Phytopathol. 40: 309-348.
Whitby, P. W., K. B. Carter, K. L. Hatter, J. J. Lipuma, and T. L. Stull. 2000.
Identification of members of the Burkholderia cepacia complex by species-species PCR.
Journ. of Clinical Microbiol. 38 (8): 2962-2965.
Whitby, P. W., L. C. Pope, K. B. Carter, J. J. Lipuma, and T. L. Stull. 2000. Speciesspecific PCR as a tool for the identification of Burkholderia gladioli. Journ. of Clinical
Microbiol. 38 (1): 282-285.
Willems, A., M. Gorr, S. Thielemans, M. Gillis, K. Kersters, and J. De Ley. 1992.
Transfer of several phytopathogenic Pseudomonas species to Acidovorax as Acidovorax
avenae subsp. avenae subsp. nov., comb. nov., Acidovorax avenae subsp. citrulli,
Acidovorax avenae subsp. cattleya, and Acidovorax konjaci. Int. J. Syst. Bacteriol. 42:
107-119.
Xie, G. L., J. C. Zheng, and T. W. Mew. 1999. Pathogenic bacteria associated with rice
seed in the subtropic and the tropic areas. Acta Agriculturae Zhjiangensis. 11 (3): 127132.
Yabuuchi, E., Y. Kosako, H. Oyaizu, I. Yano, H. Hotta, Y. Hashimoto, T. Ezaki, and M.
Arakawa. 1992. Proposal of Burkholderia gen. Nov. and transfer of seven species of the
genus Pseudomonas homology group II to the new genus, with the type species
Burkholderia cepacia (Palleroni and Holmes 1981) comb. Nov. Microbial. Immun. 36:
1251-1275.
Yabuuchi, E., Y. Kosako, I. Yano, H. Hotta, and Y. Nishiuchi. 1995. Transfer of two
Burkholderia and an Alcaligenes species to Ralstonia gen. Nov.: proposal of Ralstonia
pickettii (Ralston, Palleroni and Doudoroff 1973) comb. Nov., Ralstonia solanacearum
(Smith 1896) comb. Nov. and Ralstonia eutropha (Davis 1969) comb. Nov. Microbiol.
Immunol. 39: 897-904.
77
Yamashita, T., N. Kobayashi, N. Eguchi and Y. Saitoh. 1998. Occurrence of bacterial
disease (Burkholderia glumae, Burkholderia plantarii) in nursery boxes of rice plants in
Nagano prefecture. Proceed. of the Kanto-Tosan Plant Protect. Soc. 45: 11-13.
Yamasaki, S., et al. 1998. Effect of specific ions in agar on antibiotic production by
Burkholderia glumae. Journ. of the Faculty of Agri. Kyushu Univ. 43 (3-4): 309-314.
Yasufumi, H. 1995. Mode of action of Oxolinic acid against bacterial seedling rot of rice
caused by Pseudomonas glumae. III. Infection with P. glumae into plumules. Annals of
the Phytopath. Soc.of Jap. 61: 134-136.
Yasufumi, H. and H. Egami. 1995. Control system for bacterial grain rot of rice with
oxolinic acid and seed selection with salt solution. Ann. Phyto. Soc. Jap. 61: 405-409.
Yasunaga, T., H. Matsumoto, and Y. Shigematsu. 1985. Infection and control of bacterial
grain rot of rice. Bull. Ehime Pref. Agric. Exp. Stn. 24: 21-28.
Yoneyama, K., Y. Kono, I. Yamaguchi, M. Horikoshi, and T. Hirooka. 1998. Toxoflavin
is an essential factor for virulence of Burkholderia glumae causing rice seedling rot
disease. Annals of the Phytopath. Soc. of Jap. 64 (2): 91-96.
Yu, X-F., G. Xie, J. Swings, T. W. Mew. 2002. Relationships of different cultural
patterns of a predominant japonica rice variety and population variation of antagonistic
bacteria against rice sheath blight and the bacterial species. Chinese Journ. of Rice Sci.
16 (4): 351-355.
Zeigler, R. S., and E. Alvarez. 1987. Bacterial sheath brown rot of rice caused by
Pseudomonas fuscovaginae in Latin America. Plant Dis. 71: 592-597.
Zeigler, R. S., G. Aricapa, and E. Hoyos. 1987. Distribution of fluorescent Pseudomonas
spp. causing grain and sheath discoloration of rice in Latin American. Plant Dis. 71: 896900.
Zeigler, R. S. and E. Alvarez. 1989a. Pseudomonas species causing rice sheath rot (ShR)
and grain discoloration (GID). Int. Rice Res. Newsletter 14 (1): 26.
Zeigler, R. S. and E. Alvarez. 1989b. Grain discoloration of rice caused by Pseudomonas
glumae in LatinAmerica. Plant Dis. 73 (4): 368.
78
Zeigler, R. S. and E. Alvarez. 1989c. Differential culture medium for Pseudomonas
species causing sheath rot (ShR) and grain discoloration (GID) of rice. Int. Rice Res. Inst.
Newsl. 14: 27-28.
Ziegler, R. S. 1990. Nonfluorescent Pseudomonas strains causing rice sterility and grain
discoloration in Colombia. Int. Rice Res. Newsletter 15 (3): 28-29.
Zeigler, G. S. and E. Alvarez. 1990. Characteristics of Pseudomonas spp. causing grain
discoloration and sheath rot of rice, and associated Pseudomonas epiphytes. Plant Dis. 74
(11): 917-922.
Many papers were reviewed for this research that covered research that was
indirectly related to the research area. As these papers were not cited, but contained
information of use in future research with pathogenic bacteria on rice, they were listed in
an Appendix as “BIBLIOGRAPHY OF RELATED LITERATURE”.
79
APPENDIX
BIBLIOGRAPHY OF RELATED LITERATURE
Abed, Y., A. Davin-Regli, C. Bollet, and P. De Micco. 1995. Efficient discrimination of
mycobacterium tuberculosis strains by 16S-23S spacer region-based random amplified
polymorphic DNA analysis. J. Clin. Microbiol. 33: 1418-1420.
Aguilar, C., I. Bertani, and V. Venturi. 2003. Quorum-sensing system and stationar-phase
sigma factor (rpoS) of the onion pathogen Burkholderia cepacia genomovar I type strain,
ATCC 25416. Applied and Environmental Microbiology. 69 (3): 1739-1747.
Aunpad, R., S. P. Muench, P. J. Baker, S. Sedelnikova, W. Panbangred, N. Doukyu, R.
Aono, D. W. Rice. 2002. Crystallization and preliminary X-ray crystallographic studies
on the class II cholesterol oxidase from Burkholderia cepacia containing bound flavin.
ACTA Crystallographica Section D-Biological Crystallography. 58: 2182-2183.
Balandreau, J. A moratorium about Burkholderia use in the environment? Available:
Online: http://www.ag.uidaho.edu/bacteriology/Archives/.
Balashova, N. V., I. A. Kosheleva, N. P. Golovchenko and A. M. Boronin. 1999.
Phenanthrene metabolism by POseudomonas and Burkholderia strains. Process
Biochemistry. 35 (3-4): 291-296.
Baldani, V. L. D., J. I. Baldani, and J. Dobereiner. 2000. Inoculation of rice plants with
the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. Biology
and Fertility of Soils. 30 (5-6): 485-491.
Ballio, A., F. Bossa, L. Camoni, D. Di Giorgio, M. C. Flamand, H. Maraite, G. Nitti, P.
Pucci, and A. Scaloni. 1996. FEBS Lett 381: 213-216.
Bare, S., V. M. Coiro, A. Scaloni, A. Di Nola, M. Paci, A. L. Segre, and A. Ballio. 1999.
Conformations in solution of the fuscopeptins – Phytotoxic metabolites of Pseudomonas
fuscovaginae. European Journal of Biochemistry. 266 (2): 484-492.
Batoko, H., A. D. d’Exaerde, J. M. Kinet, J. Bouharmont, R. A. Gage, H. Maraite, and M.
Boutry. 1998. Modulation of plant pasma membrane H+ATPase by phytotoxic
lipodepsipeptides produced by the plant pathogen Pseudomonas fuscovaginae.
Biochimica et Biophysica ACTA-Biomembranes. 1327 (2): 216-226.
Batoko, H., J. Bouharmont, and H. Maraite. 1994. Inhibition of rice (Oryza-sativa L)
seedling elongation by a Pseudomonas-Fuscovaginae toxin. Euphytica. 76 (1-2): 139143.
80
Batoko, H., J. Bouharmont, J. M. Kinet, and H. Maraite. 1997. Involvement of toxins
produced by Pseudomonas fuscovaginae in aetiology of rice bacterial sheath brown rot.
Journal of Phytopathology-Phytopathologische Zeitschrift. 145 (11-12): 525-531.
Bauernfeind, A., I. Schneider, R. Jungwirth, and C. Roller. 1998. Discrimination of
burkholderia gladioli from other Burkholderia species detectable in cystic fibrosis
patients by PCR. J. Cli. Microbiol. 36: 2748-2751.
Bertilsson, S., C. M. Cavandaugh, M. F. Polz. 2002. Sequencing-independent method to
generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by
PCR with detachable primers. Appl. Environ. MIcrobiol. 68: 6077-6086.
Bevivino, A., S. Sarrocco, C. Dalmastri, S. Tabacchioni, C. Cantale, and L. Chiarini.
1998. Characterization of a free-living maize-rhizosphere population of Burkholderia
cepacia effect of seed treatment on disease suppression and growth promotion of maize.
FEMS Microbiology Ecology. 27 (3): 225-237.
Brett, P. J., D. Deshazer, and D. E. Woods. 1997. Characterization of Burkholderia
pseudomallei and Burkholderia pseudomallei-like strains. Epidemiol. Infect. 118: 137148.
Brett, P. J., M. N. Burtnick, and D. E. Woods. 2003. The wbiA locus is required for the
2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and
Burkholderia thailandensis. FEMS Microbiology Letters. 218 (2): 323-328.
Brisse, S., C. M. Verduin, D. Milatovic, A. Fluit, J. Verhoef, S. Laevens, P. Vandamme,
B. Tummler, H. A. Verbrugh, and A. Belkum. 2000. Distinguishing species of the
burkholderia cepacia complex and burkholderia gladioli by automated ribotyping. Journal
of Clin. MIcrobiol. 38: 1876-1884.
Bultreys, A., I. Gheysen, B. Wathelet, H. Maraite, and H. Hoffmann. 2003. Highperformance liquid chromatography analyses of pyoverdin siderophores differentiate
among phytopathogenic fluorescent Pseudomonas species. Appl. Environ. Microbiol. 69:
1143-1153.
Campbell, P. W., J. A. Phillips, G. J. Heidecker, M. R. Krishnamani, R. Zahorchak, T. L.
Stull. 1995. Detection of pseudomonas (Burkholderia) cepacia using PCR. Pediatr
Pulmonol. 20: 44-49.
Carpena, X., J. Switala, S. Loprasert, S. Mongkolsuk, I. Fita, and P. C. Loewen. 2002.
Crystallization and preliminary X-ray analysis of the catalase-peroxidase KatG from
Burkholderia pseudomallei. ACTA Crystallographica Section D-Biological
Crystallography. 58: 2184-2186.
Cartwright, R. and F. Lee. 2001. Bacterial panicle blight. Available: online:
http://www.uaex.edu/.
81
Cerantola, S., J-D. Bounery, C. Segonds, N. Marty and H. Montrozier. 2000.
EXopolysaccharide production by mucoid and non-mucoid strains of Burkholderia
cepacia. FEMS Microbiology Letters. 185 (2): 243-246.
Cerantola, S. and H. Montrozier. 2001. Production in vitro, on different solid culture
media, of two distinct exopolysaccharid by a mucoid clinical strain of Burkholderia
capacia. FEMS Microbiology Letters. 202 (1): 129-133.
Clode, P. E., M. E. Kaufmann, H. Malnick, and T. L. Pitt. 1999. Evaluation of three
oligonucleotide primer sets in PCR for the identification of Burkholderia cepacia and
their differentiation from Burkholderia gladioli. J. of Clin. Pathol. 52 (3): 173-176.
Chen, Z. and T. W. Mew. 1998. Relationship between the colonization, concentration and
spray timing of antagonistic bacteria and sheath blight of rice. Jiangsu J. of Agr. Sci. 14
(1): 31-35.
Cheneby, D., L. Philipport, A. Hartmann, C. Henault and J-C, Germon. 2000. 16S rDNA
analysis for characterization of denitrifying bacteria isolated from three agricultural soils.
FEMS Microbiology Ecology. 34 (2): 121-128.
Cho, Y-S., S-H. Park, C-K. Kim, and K-H. Oh. 2000 Induction of stress shock proteins
DnaK and GroEL by phenoxyherbicide 2, 4-D in Burkholderia sp. YK-2 isolated from
rice field. Curr. Microbiol. 41: 33-38.
Chung, J. W., E. Altman, T. J. Beveridge, and D. P. Speert. 2003. Colonial morphology
of Burkholderia cepacia complex genomovar III: Implications in exopolysaccharide
production, pilus expression, and persistence in the mouse. Infection and immunity. 71
(2): 904-909.
Coenye, T., E. Mahenthiralingam, D. Hernry, J. J. LiPuma, S. Laevens, M. Gillis, D. P.
Speert, and P. Vandamme. 2001. Burkholderia ambifaria sp. nov., a novel member of the
Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates. J.
Syst. Evol. Microbiol. 51: 1481-1490.
Coenye, T., B. Holmes, K. Kersters, J. R. Govan, and P. Vandamme. 1999. Burkholderia
cocovenenans (van Damme et al. 1960) Gillis et al. 1995 and Burkholderia vandii
Urakami et al. 1994 are junior synonyms of Burkholderia gladioli (Severini 1913)
Yabuuchi et al. 1993 and Burkholderia plantarii (Azegami et al. 1987) Urakami et al
1994, respectively. Int. J. Syst. Bacteriol. 49: 37-42.
Coenye, T., M. Gillis and P. Vandamme. 2000. Pseudomonas antimicrobica attafuah and
Bradbury 1990 is a junior synonym of Burkholderia gladioli (Severini 1913) Yabuuchi et
al. 1993. Int. J. of Syst. And Evol. Microbiol. 50: 2135-2139.
82
Coenye, T., T. Spilker, A. Martin, and J. J. LiPuma. 2002. Comparative assessment of
genotyping methods for epidemiologic study of Burkholderia cepacia genomovar III. J.
of Clin. Microbiol. 40 (9): 3300-3307.
Coenye, T. and J. J. LiPuma 2003. Molecular epidemiology of Burkholderia species.
Frontiers in Bioscience. 8: 55-67.
Coenye, T. and J. J. LiPuma. 2003. Population structure analysis of burkholderia cepacia
genomovar III: varying degrees of genetic recombination characterize major clonal
complexes. Microbiology. 149: 77-88.
Cottyn, B. E. Regalado, B. Lanoot, M. De Cleene, T. W. Mew, and J. Swings. 2001.
Bacterial populations associated with rice seed in the tropical environment.
Phytopathology. 91 (3): 282-292.
Derakshani, M., T. Lukow, and W. Liesack. 2001. Novel bacterial lineages at the (sub)
division level as detected by signature nucleotide-targeted recovery of 16S rRNA sgenes
from bulk soil and rice roots of flooded rice microcosms. Appl. Environ. Microbiol. 67:
623-631.
Duveiller, F. Snacken, H. Maraite, and A. Autrique. 1989. 1st Detection of Pseudomonafuscovaginae on maize and sorghum in Burundi. Plant Dis. 73 (6): 514-517.
Duveiller, E., J. L. Notteghem, P. Rott, F. Snacken, and H. Maraite. 1990. Bacterial
sheath brown rot of rice caused by pseudomonas-fuscovaginae in Malagasy. Trop. Pest
Mgnt. 36 (2): 151-153.
El Khattabi, M., C. Ockhuijsen, W. Bitter, K.-E. Jageger, and J. Tommassen. 1999.
Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the
membrane anchor. Molecular and General Genetics. 261 (4-5): 770-776.
El Khattabi, M., P. Van Gelder, W. Bitter, and J. Tommassen. 2000. Role of the lipasespecific foldase of Burkholderia glumae as a steric chaperone. J. of Biological Chemistry.
275 (35): 26885-26891.
Engelhard, M., T. Hurek, and B. Reinhold-Hurek. 2000. Preferential occurrence of
diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza
sativa in comparison with modern races. Environmental Microbiology. 2 (2): 131-141.
Estrada, P., P. Mavingui, B. Cournoyer, F. Fontaine, J. Balandreau, and J. CaballeroMellado. 2002. A N2-fixing endophytic Burkholderia sp. associated with maize plants
cultivated in Mexico. Can. J. MIcrobiol. 48: 285-294.
83
Estrada-De Los Santos, P., R. Bustillos-Cristales, and J. Caballero-Mellado. 2001.
Burkholderia, a genus rich in plant-associated nitrogen fixers with wide environmental
and geographic distribution. Appl. Environ. Microbiol. 67: 2790-2798.
Fehlner-Gardiner, C. C., T. M.-H. Hopkins and A. M. Valvano. 2002. Identification of a
general secretory pathway in a human isolate of Burkholderia vietnamiensis (formerly B.
cepacia complex genomovar V) that is required for the secretion of hemolysin and
phospholipase C activities. Microbial. Pathogenesis. 32 (5): 249-254.
Flamand, M.C., S. Pelsser, E. Ewbank, and H. Maraite. 1996. Production of syringotoxin
and other bioactive peptides by Pseudomonas fuscovaginae. Physiological and Molecular
Plant Pathology. 48 (4): 217-231.
Flore, A., S. Laevens, A. bevivinao, C. Dalmastri, S. Tabacchioni, P. Vandamme, and L.
Chiarini. 2001. Burkholderia cepacia complex: distribution of genomovars among
isolates from the maize rhizosphere in Italy. Environ. Microbiol. 3: 137-143.
Furuya, N., K. Iiyama, Y. Ueda, and N. Matsuyama. 1997. Reaction of tobacco and rice
leaf tissue infiltrated with Burkholderia glumae or B-gladioli. J. of the Faculty of Agr.
Kyushu University. 42 (1-2): 43-51.
Furuya, N., K. Iiyama, N. Shiozaki, and N. Matsuyama. 1997. Phytotoxin produced by
Burkholderia gladioli. J. of the Faculty of Agr. Kyushu University. 42 (1-2): 33-37.
Furuya, N., Y. Kushima, and N. Matsuyama. 1992. Production of antibacterial substances
by Pseudomonas-glumae. J. of the Faculty of Agr. Kyushu University. 37 (2): 149-158.
Furuya, N., U. Hiroyuki, K. Iiyama, M. Matsumoto, M. Takeshita, and Y. Takanami.
2002. Specific oligonucleotide primers baqsed on sequences of the 16S-23S rDNA spacer
region for the detection of Burkholderia gladioli by PCR. J. Gen. Plant Pathol. 68: 220224.
Galbrath, L., H. Martina, L. Jonsson, C. Rudhe and S. G. Wilkinson. 1999. Lipids and
fatty acids of Burkholderia and Ralstonia species. FEMS Microbiol. Letters. 173 (2):
359-364.
Gangadhara, K. P. and A. A. Kunhi. 2000. Protection of tomato seed germination from
the inhibitory effect of 2, 4, 5-trichlorophenoxyacetic acid by inoculation of soil with
Burkholderia cepacia AC1100. J. Agric. Food Chem. 48: 4314-4319.
Gardan, L., P. Bella, J. M. Meyer, R. Christen, P. Rott, W. Achouak, R. Samson. Int.
Syst. Evol. Microbiol. 52: 2065-2074.
Garrec, G. M-L., i. Artaud and C. Capeillere-blandin. 2001. Purification and catalytic
properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain
84
AC1100. Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Enzymology. 1547 (2): 288-301.
Gillis, M., T. Van Van, R. Bardin, M. Goor, P. Hebbar, A. Willems, P. Segers, K.
Kersters, T. Heulin, and M. P. Fernandez. 1995. Polyphasic taxonomy in the genus
Burkholderia leading to an emended description of the genus and proposition of
Burkholderia vietnamiensis sp. nov. for N inferior 2-fixing isolates from rice in Vietnam.
Int. J. of Syst. Bacteriol. 45(2): 273-289.
Graves, M., T. Robin, A. M. Chipman, J. Wong, S. Khashe, and J. M. Janda. 1997. Four
additional cases of Burkholderia gladioli infection with microbiological correlates and
review. Clin. Infect. Dis. 25:838-842.
Gronow, S., C. Noah, A. Blumenthal, B. Lindner, and H. Brade. 2003. Construction of a
deep-rough matant of Burkyholderia cepacia ATCC 25416 and characterization of its
chemical and biological properties. J. of Biological Chemistry. 278 (3): 1647-1655.
Hanzawa, S. and K. Kaji. 1991. Secondary infection of rice seeding rot in a nursery box,
caused by grain rot bacterium, Psedomonas glumae Kurita and Tabei 1967 and rot of
diseased seedling in paddy field. Ann. Rept. Plant Prot. North Japan. 42: 27-30.
Hasebe, A., S. Tsushima, and S. Iida. 1998. Isolation and characterization of IS1416 from
Pseudomonas glumae, a new member of the IS3 family. Plasmid. 39: 196-204.
Hasebe, A. and S. Iida. 2000. The novel insertion sequence IS 1417, IS1418, and IS1419
from Burkholderia glumae and their strain distribution. Plasmid. 44: 44-53.
Hayashi, K., J. Oyama, A. Kikuia, and K. Fuijj. 1997. The pool-nursery, as a countermeasure of rice bacterial seedling blight. Ann. Rept. Plant Prot. North Japan. 48: 47-49.
Henry, D. A., M. E. Campbell, J. J. LiPuma, and D. P. Speert. 1997. Identificationof
Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new
selective medium. J. Clin. MIcrobiol. 35: 614-619.
Henry, D. A., E. Mahenthiralingam, P. Vandamme, T. Coenye, and D. P. Speert. 2001.
Phenotypic methods for determining genomovar status of the Burkholderia cepacia
complex. J. Clin. Microbiol. 49: 1073-1078.
Heuer, H. and K. Smalla. 1997. Evaluation of community-level catabolic profiling using
Biolog GN microplates to microbial community changes in potato phyllosphere. J. of
Microbiological Methods. 30 (1): 49-61.
Hikichi, Y., T. Okuno, and I. Furusawa. 1994. Susceptibility of rice spikelets to infection
with pseudomonas-glumae and its population-dynamics. J. of Pest. Sci. 19 (1): 11-17.
85
Hopfl, P., W. Ludwig, K. H. Schleifer, and N. Larsen. 1989. The 23S ribosomal RNA
higher-order structure of Pseudomonas cepacia and other prokaryotes. European J.
Biochem. 185: 355-364.
Hu, F. P. and J. M. Young. 1998. Biocidal activity in plant pathogenic acidovorax,
Burkholderia, Herbaspirillum, Ralstonia and Xanthomonas spp. J. Appl. Microbiol. 84:
263-271.
Hu, F. P., J. M. Young, and M. J. Fletcher. 1998. Preliminary description of biocidal
(syringomycin) activity in fluorescent plant pathogenic Pseudomonas species. J. Appl.
Microbiol. 85: 365-371.
Iguchi, K. 1991. Growth development of rice plant suffering from bacterial seedling
blight after transplanting and the population changes of the causal agent, Pseudomonas
plantarii in rice plant. Proceedings of the Kanto-Tosan Plant Protection Society. 38: 2324.
Iiyama, K., N. Furuya, K. Hara, N. Nakashima, Y. Takanami, and N. Matsuyama. 1994.
Phytotoxin Produced by Pseudomonas-glumae Kurita-et-Tabei, a causal bacterium of the
grain and seedling rot of rice. J. of the Faculty of Agr. Kyushu University. 38 (3-4): 175181.
Iiyama, K., N. Furuya, H. Ura, and N. Matsuyama. 1998. Role of phytotoxins in the
pathogenesis of Burkholderia species. J. of the Faculty of Agr. Kyushu University. 42 (34): 289-293.
Inose, K., M. Fujikawa, T. Yaniazaki, K. Kojima, and K. Sode. 2003. Cloning and
expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase
from Burkholderia cepacia in Escherichia coli. Biochimica et Biophysica Acta-Proteins
and Proteomics. 1645 (2): 133-138.
Isogawa, Y., S. Kato, and S-H. Li. 1989. Studies on environmental factors and control
methods of bacterial grain rot of rice caused by Pseudomonas glumae Kurita et Tabei II.
REs. Bull. Aichi. Agric. REs. Ctr. 21: 85-90.
Iwai, T., H. Kaku, R. Honkura, S. Nakamura, H. Ochiai, T. Sasaki, and Y. Ohashi. 2002.
Enhanced resistance to seed-transmitted bacterial diseases in transgenic rice plants
overproducing an oat cell-wall-bound thion. Mol. Plant Microbe Interact. 15: 515-512.
Jaeger, K-E. and M. T. Reetz. 1998. Microbial lipases form versatile tools for
biotechnology. A TRENDS Guide to Proteomics II. 16 (9): 396-403.
Jaunet, T., G. Laguerre, P. Lemanceau, R. Frutos, and J. L. Notteghem. 1995. Diversity
of pseudomonas fuscovaginae and other fluorescent pseudomonads isolated from
diseased rice. Phytopathology 85 (12): 1534-1541.
86
Jaunet, T., J. L. Notteghem, and F. Rapilly. 1996. Pathogenicity process of Pseudomonas
fuscovaginae, the causal agent of sheath brown rot of rice. J. of PhytopathologyPhytopathologische Zeitschrift. 144 (9-10): 425-430.
Kadota, I. 1993. Studies on the infection cycle and control of bacterial brown stripe of
rice caused by Pseudomonas avenae. Ann. Phytopath. Soc. Japan 59: 242.
Katoh, T., Y. Fujita and H. Sakuma. 1990. Tropolone method to find rice seed infected
with bacterial seedling blight. Ann. Rept. Plant Prot North Japan. 41: 47-49.
Katsube, K. and S. Takeda. 1998. Increase in severity of bacterial seedling blight of rice
with soil drenching of fungicides. Ann. Rept. Plant Prot. North Japan. 49: 37-49.
Katsuyoshi, Y., K. Yoshiki, Y. Isamu, H. Mamoru, and H. Takashi. 1998. Toxoflavin is
an essential factor for virulence of Burkholderia glumae causing rice seedling rot disease.
Ann. of the Phyto. Soc. of Japan. 64 (2): 91-96.
Kenna, D. T., V. A. Barcus, R. J. Langley, P. Vandamme, and J. R. W. Govan. 2003.
Lack of correlation between O-serotype, bacteriophage susceptibility and genomovar
status in the Burkholderia cepacia complex. FEMS Immunology and Med. Microbiology.
35 (2): 87-92.
Knee, E. M., F. C. Gong, M. Gao, M. Teplitski, A. R. Jones, A. Foxworthy, A. J. Mort,
and W. D. Bauer. 2001. Root mucilage from pea and its utilization by rhizosphere
bacteria as a sole carbon sourc. Molecular Plant-Microbe Interactions. 14 (6): 775-784.
Koga, K., N. Furuya, Z. W. Wang, S. Wakimoto, and N. Matsuyama. 1993. Pathogenicity
of the Tn5 inserted mutant strains of Pseudomonas-glumae. J. of the Faculty of Agr.
Kyushu Univ. 37: 359-369.
Kong, H., M. Shimosaka, Y. Ando, K. Nishiyama, T. Fujii, and K. Miyashita. 2001.
Species-specific distribution of a modular family 19 chitinase gene in Burkholderia
gladioli. FEMS Microbiology Ecology. 37 (2): 135-141.
Koshino, H., Y. Kono, K. Yoneyama, and J. Uzawa. 2000. N-15 NMR study of
toxoflavin and fervenulin. Heterocycles. 52 (2): 811-817.
Kotilainen, P., J. Jalava, O. Meurman, O. P. Lehtonen, E. Rintala, O. P. Seppala, E.
Eerola, and S. Nikkari. Diagnosis of meningococcal meningitis by broad-range bacterial
PCR with cerebrospinal fluid. J. Clin. Microbiol. 36: 2205-2209.
Kreader, C. A. 1995. Design and evaluation of bacteroides DNA probes for the specific
detection of human fecal pollution. App.Environ. Microbiol. 61: 1171-1179.
Lagatolla, C., S. Skerlavaj, L. Dolzani, E. A. Tonin, C. M. Bragadin, M. Bosco, R. Rizzo,
L. Giglio and P. Cescutti. 2002. Microbiological characterization of Burkholderia
87
cepacia isolates from cystic fibrosis patients: investigation of the exopolysaccharides
produced. FEMS Microbiology Letters. 209 (1): 95-102.
Lefebre, M. D. and M. A. Valvano. 2002. Construction and evaluation of plasmid vectors
optimized for constitutive and regulated gene expression in Burkholderia cepacia
complex isolates. Applied and Environmental Microbiology. 68 (12): 5956-5964.
Leelayuwat, C., A. Romphruk, A. Lulitanond, S. Trajulsomboon and V. Thamlikitkul.
2000. Genotype analysis of Burkholderia pseudomallei using randomly amplified
polymorphism DNA (RAPD): indicative of genetic differences amongst environmental
and clinical isolates. Acta Tropica. 77 (2): 229-237.
Leu. L. S. and H. C. Yang. 1985. Distribution and survival of sclerotia of rice sheath
blight fungus, Thanate phorus cucumeris, in Taiwan. Ann. Phytopath. Soc. Japan 51: 1-7.
Li, H. Q., I. Matsuda, Y. ujise, and A. Ichiyama. 1999. Short-chain acyl-CoA-dependent
production of oxalate from oxaloacetate by burkholderia glumae, a plant pathogen which
causes grain rot and seedling rot of rice via the oxalate production. J. Biochem. (Tokyo)
126: 243-253.
LiPuma, J. J., B. J. Dulaney, J. D. McMenamin, P. W. Whitby, T. L. Stull, T. Coenye,
and P. Vandamme. 1999. Development or rRNA-based
pcr assays for identification of Burkholderia cepacia complex isolates recovered from
cystic fibrosis patients. J. Clin. Microbiol. 37: 3167-3170.
Liu, P. Y., Z. Y. Shi, Y. J. Lau, B. S. Hu, J. M. Shyr, W. S. Tsai, Y. H. Lin, and C. Y.
Tseng. 1995. Comparson of different PCR approaches for characterization of
Burkholderia (Pseudomonas) cepacia isolates. J. Clin. MIcrobiol. 33: 3304-3307.
Mattos, K. A., C. Jones, N. Heise, J. O. Previato, and L. Mendonca-Previato. Structure of
an acidic exopolysaccharide produced by the diazotrophic endophytic bacterium
Burkholderia brasiliensis. European J. of Biochemistry. 268 (11): 3174-3179.
Meyer, J. M., V. T. Van, A. Stintzi, O. Berge, and G. Winkelmann. 1995. Ornibactin
production and transport-properties in strains of Burkholderia-vietnamiensis and
Burkholderia-cepacia (formerly Pseudomonas-cepacia). Biometals. 8 (4): 309-317.
Meyer, J. M., V. A. Geoffroy, N. Baida, L. Gardan, D. Izard, P. Lemanceau, W.
Achouak, N. J. Palleroni. 2002. Siderophore typing, a powerful tool for the identification
oflfuorescent and nonfluorescent pseudomonads. Appl. Environ. Microbiol. 68: 27452753.
Michel, V. V., G. L. Hartman, and D. J. Midmore. 1996. Effect of previous crop on soil
populations of Burkholderia solanacearum, bacterial wilt, and yield of tomatoes in
Taiwan. Plant disease. 80 (12): 1367-1372.
88
Miche, L. and J. Balandreau. 2001. Effects of rice seed surfaces sterilization with
hypochlorite on inoculated Burkholderia vietnamiensis. Applied and Environmenal
Microbiology. 67 (7): 3046-3052.
Mizuno, A., S. Tsushima, I. Kadota, and K. Nishiyama. 1995. A cloned DNA probe for
detection of Pseudomonas gladioli. Ann. Phytopathol. Soc. Japan. 61: 22-26.
Nacamulli, C., A. Bevivino, C. Dalmastri, S. Tabacchioni, and L. Chiarini. 1997.
Perturbation of maize rhizosphere microflora following seed bacterization with
Burkholderia cepacia MCI 7. FEMS Microbiology Ecology. 23 (3): 183-193.
Nagamatsu, T., H. Yamasaki, T. Hirota, Y. Kido, M. Shibata, and F. Yoneda. 1992.
Synthesis of 6-phenyl analogs of toxoflavin and their 4-oxides and antimicrobial
activities of toxoflavin and its analogs. J. of Pharmacobio-Dynamics. 15 (7): 93.
Nakata, P. A. 2002. The generation of a transposon-mutagenized Burkholderia glumae
library to isolate novel mutants. Plant Science. 162 (2): 267-271.
Nzula, S, P. Vandamme, and J. R. W. Govan. 2002. Influence of taxonomic status on the
in vitro antimicrobial susceptibility of the Burkholderia cepacia complex. J. of
Antimicrobial Chemotherapy. 50: 265-269.
Oh, C. J., H. B. Kim, and C. S. An. 2003. Molecular cloning and complementation
analysis of nifV gene from Frankia EuIK1 strain. Molecules and cells. 15 (1): 27-33.
Ohta, K. and R. Tei. 1994. Chemical control of bacterial seedling blight and bacterial
grain rot of rice in nursery stage. Proceedings of the Kanto-Tosan Plant Protection
Society. 41: 9-11.
Ohta, K. 1995. A simple detection method for seed infected with bacterial seedling blight
pathogen. Proceedings of the Kanto-Tosan Plant Protection Society. 42: 15-17.
Ohta, K. 1995. Control of bacterial seedling blight of rice by vacuum soaking method.
Proceedings of the Kanto-Tosan Plant Protection Society. 42: 19-21.
Pallud, C., V. Viallard, J. Balandreau, P. Normand, and G. Grundmmann. 2001.
Combined use of aspecific probe and PCAT medium to study Burkholderia in soil. J.
Microbiological Methods. 47 (1): 25-34.
Parke, J. L. 1998. Burkholderia cepacia: Friend or Foe? Available: online:
http://www.apsnet.org/online/feature/BurkholderiaCepacia/.
Parke, J. L. and D. Gurian-Sherman. 2001. Diversity of the Burkholderia cepacia
complex and implications for risk assessment of biological control strains. Annu. Rev.
Phytopathol. 39: 225-258.
89
Parsons, Y. N., K. J. Glendinning, V. Thornton, B. A. Hales, C. A. Hart, and C.
Winstanley. 2001. A putative type III secretion gene cluster is widely distributed in the
Burkholdeia cepacia complex but absent from genomovar I. FEMS Microbiol. Letters.
203 (1): 103-108.
Perez-Prat, E. and M. M. van L. Campagne. 2002. Hybrid seed production and the
challenge of propagating male-sterile plants. Trends in Plant Science. 7 (5): 199-203.
Pitt, T. L., S. Trakulsomboon and D. A. B. Dance. 2000. Molecular phylogeny of
Burkholderia pseudomallei. Acta Tropica. 74 (2-3): 181-185.
Porteous L. A., F. Widmer, and R. J. Seidler. 2002. Multiple enzyme restriction fragment
length polymorphism analysis for high resolution distinction distinction of Pseudomonas
(sensu stricto) 16S rRNA genes. J. Microbiol Methods. 51: 337-348.
Putra, S. R., A. Disch, J-M. Bravo, and M. Rohmer. 1998. Distribution of mevalonate and
glyceraldehydes 3-phosphate/pyruvate routes for isoprenoid biosynthesis in some Gramnegative bacteria and mycobacteria. FEMS Microbiology Letters. 164 (1): 169-175.
Rathi, P., V. K. Goswami, V. Sahai, and R. Gupta. 2002. Statistical medium optimization
and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor.
J. of Applied Microbiology. 93 (6): 930-936.
Reetz, M. T. and K-E. Jaeger. 1998. Overexpression, immobilization and
biotechnological application of Pseudomonas lipases. Chemistry and Physics of Lipids.
93 (1-2): 3-14.
Reiter, B., A. Glieder, D. Talker, and H. Schwab. 2000. Cloning and characterization of
EstC from Burkholderia gladioli, a novel-type esterase related to plant enzyme. Appl.
Microbiol. Biotechnol. 54: 778-785.
Rehm, B. H. A. 2002. Molecular characterization of the poly (3-hydroxybutyrate) (PHB)
SYNTHASE FROM Ralstonia eutropha: in vitro evoluation, site-specific mutagenesis
and development of a PHB synthase protein model. Proteins and Proteomics. 1594 (1):
178-190.
Richardson, J., D. E. Stead, J. G. Elphinstone, and R. H. Coutts. 2002. Diversity of
Burkholderia isolates from voodland rhizosphere environments. J. Appl. Microbiol. 93:
616-630.
Riedel, K., C. Arevalo-Ferro, G. Reil, A. Gorg, F. Lottspeich, and L. Eberl. 2003.
Analysis of the quorum-sensing regulon of the opportunistic pathogen Burkolderia
capacia H111 by proteomics. Electrophoresis. 24 (4): 740-750.
Rosenau, F. and K-E. Jaeger. 2000. Bacterial lipases from Pseudomonas: Regulation of
gene expression and mechanisms secretion. Biochimie. 82 (11): 1023-1032.
90
Rosenstein, R. and F. Gotz. 2000. Staphylococcal lipases: Biochemical and molecular
characterization. Biochimie. 82 (11): 1005-1014.
Rott, P., J. L. Notteghem, and P. Frossard. 1989. Identification and characterization of
Pseudomonas-fuscovaginae, the causal agent of bacterial sheath brown rot of rice, from
Madagascar and other countries. Plant Disease. 73 (2): 133-137.
Rott, P., J. Honegger, J. L. Notteghem, and S. Ranomenjanahary. 1991. Identification of
Pseudomonas-fuscovaginae with biochemical, serological, and pathogenicity tests. Plant
Disease. 75 (8): 843-846.
Rumbaugh, K. P., J. A. Griswold, and A. N. Hamood. 2000. The role of quorum sensing
in the in vitro virulence of Pseudomonas aeruginose. Microbes and Infection. 2 (14):
1721-1731.
Ryley, H. C., L. Millar-Jones, A. Paull, and J. Weeks. 1995. Characterization of
Burkholderia cepacia from cystic fibrosis patients living in Wales by PCR ribotyping. J.
Med. Microbiol. 43: 436-441.
Salles, J. F., F. A. De Souza, and J. D. van Elsas. 2002. Molecular method to assess the
diversity of Burkholderia species in environmental samples. Appl. Environ. Microbiol.
68: 1595-1603.
Schlacher, A., T. Stanzer, I. Osprian, M. Mischitz, E. Klingsbichel, K. Faber, and H.
Schwab. 1998. Detection of a new enzyme for stereoselective hydrolysis of linalyl acetate
using sim. Plate assays for the characterization of cloned esterase from Burkholderia
gladioli. J. of Biotechnology. 62 (1): 47-54.
Schmitz, F. J., C. R. Mackenzie, B. Hofmann, J. Verhoef, M. Finken-Eigen, H. P. Heinz,
and K. Kohrer. 1997. Specific information concerning taxonomy, pathogenicity and
methicillin resistance of staphylococci obtained by a multiplex PCR. J. Med. Microbiol.
46: 773-778.
Schloter, M. M. Lebuhn, T. Heulin, and A. Hartmann. 2000. Ecology and evolution of
bacterial microdiversity. FEMS. Microbiology Review. 24 (5): 647-660.
Segonds, C., T. Heulin, N. Marty, and G. Chabanon. 1999. Differentiationof
Burkholderia species by PCR-restriction fragment length polymorphism analysis of the
16S rRNA gene and application to cystic fibrosis isolates. J. Clin. Microbiol. 37: 22012208.
Segonds, C. and G. Chabanon. 2001. Burkholderia cepacia: dangers of a phytopathogen
organism for patients with cystic fibrosis. Ann. Biol. Clin. 59: 259-269.
91
Sermswan, R. W., S. Wongratanacheewin, S. Trakulsomboon, and V. Thamlikitful. 2001.
Ribotyping of Burkholderia psedomallei fromclinical and soil isolates in Thailand. Acta
Tropica. 80 (3): 237-244.
Silva, L. F., J. G. C. Gomez, M.S. Oliveira, and B. B. Torres. 2000. Propionic acid
metabolism and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB3HV) production
by Burkholderia sp. J. of Biotechnology. 76 (2-3): 165-174.
Simpson, I. N., J. Finlay, D. J. Winstanley, N. Dewhurst, J. W. Nelson, S. L. Butler, and
J. R. W. Govan. 1994. Multi-resistance isolate possessing characteristics of both
Burkholderia (Pseudomonas) cepacia and Burkholderia gladioli from patients with cystic
fibrosis. J. Antimicrobial Chemotherapy. 34: 353-361.
Smith-Vaughan, H. C., D. Gal, P. M. Lawrite, C.Winstanley, K. S. Sriprakash, and B. J.
Currie. 2003. Ubiquity of putative type III secretion genes among clinical and
environmental Burkholderia pseudomallei isolates in Northern Australia. J. of Clin.
Microbiol. 41 (2): 883-885.
Smith, M. D., V. Wuthiekanun, A. L. Walsh, and N. J. White. 1995. Quantitative
recovery of Burkholderia pseudomallei from soil in Thailand. Transactions of the Royal
Society of Tropical Medicine and Hygiene. 89 (5): 488-490.
de Souza, J. T. and J. M. Raaijmakers. 2003. Polymorphisms within the prnD and pltC
genes from pyrrolnitrin and pyoluteorin-producing Pseudomonas and Burkholderia spp.
FEMS Microbiol. Ecology. 43 (1): 21-34.
Sprague, L. D., G. Zysk, R. M. Hagen, H. Meyer, J. Ellis, N. Anuntagool, Y. Gauthier,
and H. Neubauer. 2002. A possible pitfall in the identification of Burkholderia mallei
using molecular identification systems based on the sequence of the flagellin flic gene.
FEMS Immunology and Medical Microbiology. 34 (3): 231-236.
Stead, D. E. 1992. Grouping of plant-pathogenic and some other Pseudomonas spp. by
using cellular fatty acid profiles. International Journal of Systematic Bacteriology. 42 (2):
281-295.
Sthapit, B. R., P. M. Pradhanang, and J. R. Witcombe. 1995. Inheritance and selection of
field-resistance to sheath brown-rot disease in rice. Plant Disease. 79 (11): 1140-1144.
Stull, T. L., J. J. LiPuma and T. D. Edlind. 1988. A broad-spectrum probe for molecular
epidemiology of bacteria: ribosomal RNA. The J. of Infectious Diseases. 157 (2): 280285.
Takahash, C. 1993. Effect of Autoclave of nursery bed soil on occurrence of bacterial
seedling blight, Pseudomonas plantarii, vergara et al. Ann. Rept. Plant Prot. North Japan.
44: 14-15.
92
Takeuchi, T. 1994. A method for testing the effect of seed disinfection for bacterial
seedling blight of rice. Ann. Rept. Plant Prot. North Japan. 45: 21-26.
Takeuchi, T. 1994. Influence of cultural conditions in nursery bed on occurrence of
bacterial seedling blight of rice. Ann. Rept. Plant Prot. North Japan. 45: 17-20.
Takikawa, Y., M. Jojima, S. Adachi, M. Kobayashi, T. Jumakura and Y. Inoue. 2000.
Cloning and characterization of hrp genes from Burkholderia glumae, Acidovorax
avenae, Plantoea agglomerans PV. millettiae and Pseudomonas fluorescens. Available:
online: http://www.bspp.org.uk/icpp98/.
Thao, M. L., P. J. Gullan, and P. Baumann. 2002. Secondary (proteobacteria)
endosymbionts infect the primary (proteobacteria) endosymbionts of mealybugs multiple
times and coevolve with their hosts. Appl. And Enviro. Microbiol. 68 (7): 3190-3197.
Tan, M-W. 2002. Cross-species infections and their analysis. Annu. Rev. Microbiol. 56:
539-565.
Tomich, M., A. Griffith, C. A. Herfst, J. L. Burns and C. D. Mohr. 2003. Attenuated
virulence of a Burkholderia cepacia type III secretion mutant in a murine model of
infection. Infection and Immunity. 73 (3): 1405-1415.
Tsushima, S., C. Narimatsu, A. Mizuno, and R. Kimura. 1994. Cloned DNA probes for
detection of Pseudomonas glumae causing bacterial grain rot of rice. Ann. Phytopath.
Soc. Japan. 60: 576-584.
Tsushima, S., H. Naito, and M. Koitabashi. 1995. Forecast of yield loss suffered from
bacterial grain rot of rice in paddy fields by severely diseased panicles. Ann. Phyto. Soc.
Japan. 61: 419-424.
Tungpradabkul, S., S. Wajanarogana, S. Tunpiboonsak, and S. Panyim. 1999. PCR-RFLP
analysis of the flagellin sequences for identification of Burkholderia pseudomallei and
Burkholderia cepacia from clinical isolates. Mol. Cell Probes. 13: 99-105.
Ulett, G. C., B. J. Currie, T. W. Clair, M. Mayo, N. Ketheesan, J. Labroc, D. Gal, R.
Norton, C. A. Smith, J. Barnes, J. Warner, and R. G. Hart. 2001. Burkholderia
pseudomallei virulence: definition, stability and association with clonalies. Microbes and
Infection. 3 (8): 621-631.
Van Ovevelen, S., R. De Wachter, P. Vandamme, E. Robbrecht, and E. Prinsen. 2002.
Identification of the bacterial endosymbionts in leaf galls of Psychotria (Rubiaceae,
angiosperms) and proposed of ‘Candidatus Burkholderia Kirkii’ sp. nov. International J.
of Syst. And Evolutionary Microbiology. 52 (6): 2023-2027.
Van Pelt, C., C. M. Verduin, W. H. F. Goessens, M. C. Vos, B. Tummler, C. Segonds, F.
Reubsaet, H. Verbrugh, and A. Van Belkum. 1999. Identification of Burkholderia spp. in
93
the clinical microbiology laboratory: comparison of conventional and molecular methods.
J. of Clin. Microbio. 37: 2158-2164.
Van, V. T., O. Berge, S. N. Ke, J. Balandreau, and T. Heulin. 2000. Repeated beneficial
effects ofrice inoculationwith a strain of Burkholderia vietnamiensis on early and late
yield components in low fertility sulphate acid soils of Vietnam. Plant and Soil. 218 (12): 273-284.
Van, V. T., O. Berge, J. Balandreau, S. N. Ke, and T. Heulin. 1996. Isolation and
nitrogenase activity of Burkholderia vietnamiensis, a nitrogen-fixing bacterium
associated with rice (Oryza sativa L) on a sulphate acid soil of Vietnam. Agronomie. 16
(8): 479-491.
Vandamme, P., D. Henry, T. Coenye, S. Nzula, M. Vancanneyt, J. J. LiPuma, D. P.
Speert, J. R. W. Govan, and E. Mahenthiralingam. 2002. Burkholderia antina sp. nov. and
Burkholderia pyrrocinia, two additional Burkholderia cepacia complex bacteria, may
confound results of new molecular diagnostic tools. FEMS Immunology and Medical
Microbiology. 33 (2): 143-149.
Vandamme, P., E. Mahenthiralingam, B. Holmes, T. Coenye, B. Hoste, P. De Vos, D.
Henry, and D. P. Speert. 2000. Identification and population structure of Burkholderia
stabilis sp. nov. (formerly Burkholderia cepacia genomovar IV). J. of Clin. Microbiol. 38
(3): 1042-1047.
Vandamme, P., D. Henry, T. Coenye, S. Nzula, M. Vancanneyt, J. J. LiPuma, D. P.
Speert, J. R. Govan, and E. Mahenthiralingam. 2002. Burkholderia anthina sp. nov. and
Burkholderia pyrrocinia, two additional Burkholderia cepacia complex bacteria, may
confound results of new molecular diagnostic tools. FEMS Immunol. Med. Microbiol.
33: 143-149.
Vandamme, P., J. Goris, W. M. Chen, P. de Vos, and A. Willems. 2002. Burkholderia
tuberum sp. nov. and Burkholderia phymatum sp. nov., nodulate the roots of tropical
legumes. Systematic and Applied Microbiology. 25 (4): 507-512.
Vandamme, P., B. Holmes, T. Coenye, J. Goris, E. Mahenthiralingam, J. J. LiPuma, and
J. R. W. Govan. 2003. Burkholderia cenocepacia sp. nov.- a new twist to an old story.
Research in Microbiology. 154 (2): 91-96.
Vandamme, P., B. Pot, M. Gillis, P. de Vos, K. Kersters, and J. Swings. 1996. Polyphasic
taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev., 60 (2): 407438.
Vermis, K., C. Vandekerckhove, H. J. Nelis, and P. A. R. Vandamme. 2002. Evaluation
of restriction fragment length polymorphism analysis of 16S rDNA as a tool for
genomovar characterization within the Burkholderia cepacia complex. FEMS Microbiol.
Letters. 214 (1): 1-5.
94
Viallard, V., I. Poirier, B. Cournoyer, J. Haurat, S. Wiebkin, K. Ophel-Keller, and J.
Balandreau. Burkholderia graminis sp. nov., a rhizospheric Burkholderia species and
reassessment of [Pseudomonas] phenazinium, [Pseudomona] pyrrocinia and
[Pseudomonas] glathei as Burkholdeia. Int. J. Syst. Bacteriol. 48 (2): 549-563.
Wang, Z. Z., M. S. Ma, and R. Wang. 1996. Enhanced vasocontraction of rat tail arteries
by toxoflavin. British J. of Pharmacology. 117 (2): 293-298.
Weller, D. M. 2002. Microbial populations responsible for specific soil suppressiveness
to plant pathogens. Annu. Rev. Phytopathol. 40: 309-348.
Whitby, P. W., H. L. Dick, P. W. Campbell, D. E. Tullis, A. Matlow, and T. L. Stull.
1998. Comparison of culture and PCR for detection of Burkolderia cepacia in sputum
samples of patients with cystic fibrosis. J. Clin. Microbiol. 36: 1642-1645.
Whitby, P. W., L. C. Pope, K. B. Carter, J. J. LiPuma, and T. L. Stull. 2000. Speciesspecific PCR as a tool for the identification of Burkholderia gladioli. J. Clin. Microbiol.
38: 282-285.
Whitby, P. W., K. B. Carter, K. L. Hatter, J. J. LiPuma, and T. L. Stull. 2000.
Identification of members of the Burkholderia cepacia complex by species-specific PCR.
J. Clin. Microbiol. 38: 2962-2965.
Winstanley, C., B. A. Hales, J. A. Morgan, M. J. Gallagher, S. D. Puthucheary, M. F.
Cisse, and C. A. Hart. 1999. Analysis of flic variation among clinical isolates of
Burkholderia cepacia. J. Med. Microbiol. 48: 657-662.
Woo, P. C., G. K. Woo, S. K. Lau, S. S. Wong, and K. Yuen. 2002. Single gene target
bacterial identification. groEL gene sequencing for discriminating clinical isolates of
Burkholderia pseudomallei and Burkholderia thailandensis. Diagn. Microbiol. Infect. Dis.
44: 143-149.
Yamasaki, S., N. Furuya, and N. Matsuyama. 1998. Effect of specific ions in agar on
antibiotic production by Burkholderia glumae. J. of the Faculty of Agr. Kyushu
University. 42 (3-4): 309-314.
Yamashita, T., N. Kobayashi, N. Eguchi, and Y. Saitoh. 1998. Occurrence of Bacterial
disease in nursery boxes of rice plants in Nagano prefecture. Proceedings of the KantoTosan Plant Protection Society. 45: 11-13.
Yao, F., H. Zhou, and T. G. Lessie. 2002. Characterization of N-acyl homoserine lactone
overproducing mutants of Burkholderia multivorans ATCC 17616. FEMS Microbiol.
Letters. 206 (2): 201-207.
95
Yuanbo, D., C. J. Zheng, C. S. Rong, S. X. Xea, and F. C. Tai. 1983. A technique for the
detection of Xanthomonas campestris pv. oryzae using bacteriophage. Seed Sci. &
Technol. 11: 579-582.
96
VITA
Xianglong Yuan was born on Juan 12, 1962, in Shenyang, Liaoning, P. R., China, where
he accomplished his elementary and high school education. In 1985, he enrolled in the
Department of Biology at the University of Liaoning. He received a Bachelors of Science honors
degree in biology in 1985. Subsequently, he was employed by the Institute of Environment
Protection at Shenyang. In 1987, he returned to school and enrolled as a M.S graduate student in
the Department of Plant Physiology and Biochemistry in the Division of Basic Science (currently
called the College of Biotechnology) at Shenyang Agriculture University. He received a M.S
degree in July, 1990. Upon graduation, he was employed by the Division of Basic Science in the
College of Traditional Chinese Medicine at Liaoning as a biochemistry lecturer until 1996.
Subsequently, he enrolled again as a graduate student in the plant physiology Ph.D. program of
the Biology Department at South China Normal University until 1998.
In the fall of 2001, he transferred to the Department of Plant Pathology and Crop
Physiology at Louisiana State University in Baton Rouge, Louisiana as a graduate student
to pursue a Masters degree under the guidance of Dr. M. C. Rush.
97