1. BACTERIAL PANICLE BLIGHT:
CAUSES AND SUGGESTED
CONTROLL MEASURES
Milton C. Rush, Donald E. Groth, Jong Ham,
And R. Nandakumar
Louisiana State University

2. Rice
Ri produced i th southern
d d in the th
United States has a long history of
loss to panicle blighting of unknown
etiology. Epidemics of panicle blight
occurred during 1995 and 1998, years
of record high temperatures, with
y
yield losses in some fields estimated to
be as high as 40%. Significant losses
were also experienced in Louisiana
during
d i 2000 and 2010 b th years of
d 2010, both f
unusually high temperature.

3. Panicle blighting had been attributed to
g g
abiotic factors including high
temperatures, water stress, or toxic
p
chemicals near the root zone, but in
1996-97 the bacterial plant p
p pathogen
g
Burkholderia glumae (formerly
Pseudomonas glumae) was identified
g )
as a cause of panicle blighting in the
southern United States. This
bacterium was first described from
Japan as the cause of g
p grain rottingg
and seedling blighting in 1956.

4. PATHOGENS
• FURTHER STUDIES INDICATED THAT TWO
PLANT PATHOGENIC BACTERIA CAUSED THE
EPIDEMICS OF PANICLE BLIGHTING
• Burkholderia glumae
– SEEDBORNE
• Burkholderia gladioli
g
– SEEDBORNE
– SOILBORNE

5. Bacterial Panicle Blight
Inoculated Non inoculated
Non-inoculated

6. SEVERE BPB

7. More than 400 isolates of the two pathogens
were isolated from diseased plants collected across
the southern United States rice area.
The two pathogens were identified by growth
on selective media, BiologTM, Cellular fatty acid
g y
analysis, and PCR. The B. glumae pathogen was
determined to be the same AS an ATTC isolate of
the pathogen f
h h from JJapan, which was fi reported
hi h first d
in 1956 as causing grain and seedling rot on rice.

8. Burkholderia glumae

9. PCR analysis of DNA isolated from B glumae (top) and B gladioli
B. B.
(bottom) strains with their respective species specific primers.
Theexpected size of 400 and 300 bp fragments were indicated by arrows.
Lane 1, 100 bp ladder; lane 2, positive control (top)-B. glumae (ATCC
(top) B.
33617) and (bottom) B. gladioli (ATCC 19302); lane 3, negative control-
(top) B. gladioli (ATCC 19302) and (bottom) B. glumae (ATCC 33617);
lanes 3-12, B. glumae isolates from infected rice grains (top); lanes 4-12,
B. gladioli strains from infected rice grains (bottom).
1 2 3 4 5 6 7 8 9 10 11 12

10. Bacterial Panicle Blight in Panama
We also observed the disease on rice
in Panama in 2002 and 2005. I
received samples in 2006 from which
p
we isolated both B. glumae and B.
gladioli based on PCR and other
identification procedures.

11. The disease was later reported from other
Asian countries and Latin America. B. glumae
p
produces the p y
phytotoxin, toxoflavin, which is
, ,
essential for its virulence and strains that lack
toxin production usually become avirulent.
The
Th symptoms of b t i l panicle blight
t f bacterial i l bli ht
include seedling blight, sheath rot lesions on the
flag leaf
flag-leaf sheath, and panicle blighting with
significant yield losses. Specific leaf sheath
symptoms include vertical lesions with gray
centers surrounded b a d k reddish b
t d d by dark ddi h brown
margin.

12. This di
Thi disease is characterized as
i h t i d
having upright, straw-colored panicles
containing florets with a darker base
and a reddish-brown line (margin of
lesion) across the floret between the
darker
d k area and th straw-colored area,
d the t l d
resulting in abortion of the kernel
before it fills. The severely affected
y
panicles remain upright, as the grain
does not fill. The term “panicle blight”
has been used in the United States for
more than 50 years and BPB has been
retained as the name for the disease in
this country.

13. Bacterial Panicle Blight

14. BACTERIAL PANICLE BLIGHT
SPRAY INOCULATED – 106 CFU / ML (quorum sensing)

15. Effect of Temperature on Growth of
BPB pathogens
Effect of temperature on the growth of
B. glumae (A) and B. gladioli (B) strains.
Each line represents g
p growth of a single
g
strain after 48 h of incubation in KBB. Each
strain was tested with three replicates. Ten
strains were tested for each species (b d
i df h i (based
on PCR and fatty acid identification).
Strains were isolated from rice panicle
collections showing symptoms of bacterial
panicle blight from Louisiana, Texas, and
Louisiana Texas
Arkansas.

16. EFFECT OF TEMPERATURE ON
GROWTH OF Burkholderia glumae
and Burkholderia gladioli STRAINS
1 .8 1 .8
1 .6 1 .6
1 .4 1 .4
nsity at 600 nm
1 .2 1 .2
1 1
Optical den
0 .8 0 .8
0 .6 0 .6
0 .4 0 .4
0 .2 0 .2
0 0
30 35 40 45 50 30 35 40 45
T e m p e r atu r e C T e m p e r atu r e C
Burkholderia glumae strains Burkholderia gladioli strains

17. The temperature optima
ranged b t
d between 38 and 40°C
d
(
(100-104F) for B. glumae and
) g
35 and 37°C (95-99F) for B.
gladioli. Th
l di li These results were
lt
confirmed with repeated
experiments.

18. Bacterial Panicle Blight in the
U.S. in 2010
TEMPERATURES were in the range of
90-100F (32-38C) during the day and in
( ) g y
the 80s (27-29C) at night in Louisiana
and Arkansas in 2010. These were new
records for night temperature (37
states). Bacterial blighting was severe in
) g g
both states according to Extension
Specialists with y
p yield losses to 50% in
some fields.

19. NIGHT TEMPERATURES
From our observations, it
observations
appears that extended high
pp g
temperatures at night seem to
be
b an iimportant factor in BPB
t tf t i
disease development.
p

20. Quorum Sensing
This is a type of decision-making
decision making
process used by decentralized groups
to coordinate behavior. Many species
of bacterial pathogens use quorum
sensing to coordinate their gene
expression according to the local
i di t th l l
density of their p p
y population.

21. A variety of diff
i t f different
t
molecules can be used as
signals. A common class of
signaling molecules in Gram-
i li l l i G
negative bacteria, such as B.
glumae and B. gladioli, is
N-Acyl Homoserine Lactones
(AHL)

22. Activation of the receptor induces
the up regulation of other specific g
p g p genes,
,
causing all of the cells to begin
transcription at approximately the same
p pp y
time. This includes the turning on of the
p
pathogen attack g
g genes. Optimum
p
temperatures for growth means that the
bacterial population reaches the
p p
threshold population to turn on these
g
genes more q quickly.
y

23. Virulence Factors Produced By Burkholderia
y
glumae in Response to Quorum Sensing
• Known: toxoflavin toxin, lipase, flagella
formation (QsmR), catalase (KatG)
(Q ), ( )
• Possible factors: type III secretion system,
extracellular polysaccharides
t ll l l h id

24. Temperature and Quorum Sensing: Relationship
to BPB Disease Development
It is known that the production of the major
virulence factors of B glumae; toxoflavin lipase
B. toxoflavin,
and flagella, are dependent on the quorum-
sensing system mediated by AHL signal
molecules. S Several virulent and avirulent B.
i i
glumae strains were tested for their ability to
produce AHL signal molecules using an AHL- AHL
biosensor strain, Chromobacterium violaceum
CV026. Interestingly, all the strains that produce
none of th virulence factors tested were d f ti
f the i l f t t t d defective
in AHL-signal biosynthesis, while the strains that
p
produce at least one of the major virulence
j
factors showed AHL-positive phenotypes.

25. Effect of loss of quorum Sensing Signals
in Avirulent Strains of B. glumae
Virulence of the B. glumae strains was
closely related to their ability to produce
various virulence factors Interestingly all
factors. Interestingly,
the confirmed avirulent strains were
defective in multiple virulence factors and
most of them lost their ability to produce
acyl-homoserine lactone (AHL) quorum-
sensing signals implying that mutation in
global regulatory system(s) for the virulence
factors is the major cause of the occurrence
of avirulent B. glumae strains in nature
f i l i i

26. Testing for Virulence
Onion scale inoculation Inoculation of rice

27. Flagella Production
Virulent Avirulent

28. Toxoflavin Toxin Production
No
N toxoflavin
fl i Toxoflavin
T fl i (yellow)

29. Inoculating Entries in the BPB Nursery

30. Three Row Plot in the BPB Nursery
Center Row Inoculated

31. Susceptible Breeding Line
S tibl B di Li

32. LM-1: Resistance Source for BPB Developed by
D.E.
D E Groth using Cobalt Irradiation of Var
Var.
Lemont

33. Susceptible Commercial Var.
Cocodrie

34. Crossing to Transfer Resistance
g
Crosses with Resistant Entries
LM –1, AB 647, LR 2065
Nipponbare,
Nipponbare Jupiter
Crosses were made with the susceptible varieties
CCDR, CPRS, and FRNS to study inheritance
of the resistance in these materials.
Results

35. SOURCES OF RESISTANCE TO BPB
1.
1 JUPITER – U S MEDIUM GRAIN VARIETY
U.S. VARIETY,
STUDIES UNDERWAY BY DR. JONG HAM
AND HIS GRADUATE STUDENTS, ,
LOUISIANA STATE UNIVERSITY
2. NIPPONBARRE, Teqing, LR 2065, LM-1, US
HYBRED VARIETIES, OTHER MATERIALS

36. Effect of B. glumae on the Yield Potential of Rice
B
Difference between sprayed and unsprayed p
p y p y plots

37. Effects of Panicle Blight on Selected
Commercial Varieties - 2005
Yield (lb/A at 12% Moisture) Difference
Varieties (lb/A at 12%
Non- Inoculated moisture)
inoculated
i l t d
Yield Yield
Rating Rating
Cocodrie 8047 7001 - 1047ns
0.8 7.3
Jupiter 10,168 9731 - 437ns
0.5
05 3.3
33
Trenasse 8338 6687 - 1651**
2.3 8.3
Bengal 8260 6262 - 1978**
2.7 8.7

38. Effects of Soilborne B. gladioli
on Yield of Bengal Rice
Soil strains sprayed Yield (lb/A at
Rating 12 % Moisture)
Moist re)
Non-inoculated (Healthy control) 2.7 8260 bc
S-10 (Soil B. gladioli) 3.7 7666 b
223 gr-1 (Grain B. gladioli) 3.3 8659 bc
3S4 (Soil B. gladioli) 2.7 8788 bc
3S5 (Soil B gladioli)
(S il B. l di li) 37
3.7 8854 c
S15 (Soil B. gladioli) 4.3 8822 bc
ATCC B. gladioli 4.3 8609 bc
336gr-1 B. glumae 8.7 6282 a

39. Chemical Control of BPB
Oxolinic acid (Starner) is the only
effective chemical control available
for this pathogen when used as a
spray, however, oxolinic acid-
resistant B. glumae strains have been
isolated from rice in Japan and
oxolinic acid is not labeled for use on
rice in the United States
States.

40. QUARANTINE
In this context use of pathogen free
context, pathogen-free
seeds is an important practice to
reduce or manage th i id
d the incidence of
f
BPB. Therefore, it was essential to
develop rapid, sensitive and
inexpensive methods for identifying
p y g
and quantifying the levels of B. glumae
in certified seeds.

41. Testing Seeds
• We d l d th d f t ti
W developed methods for testing
seed with PCR and Real-Time PCR
a. Indirect method
b.
b Direct method
• Semi-selective media (S-Pg and
CCNT)

42. CONTROLLING BPB
1. OXOLINIC ACID/STARNER (JAPAN)
2. COPPER FUNGICIDES ????
3.
3 SEED TREATMENT
4. TREATMENT OF SEED IN PRE-
SPROUTING WATER
5. HEAT TREATMENT OF SEED
6. DISEASE RESISTANCE
7.
7 QUARANTINE

43. HEAT TREATMENT OF RICE
SEEDS TO CONTROL BPB
• TREATMENT OF DRY SEED FOR 5-6 DAYS
AT 65°C
• WET TREATMENT OF SEEDS AT 62°C FOR
7.5-10 MINUTES (DEPENDS ON VARIETY
RESPONSE – GERMINATION)
NOTE: COOL SEEDS BY PLACING IN COOL
WATER IMMEDIATELY, REMOVE AND
DRY

44. TREATING PRE-SPROUTED
SEEDS
3
1
2 4

45. Stand counts

46. Trial II -
Beginning of Heading

47. Diseased Panicle
INFECTION FROM INFECTED SEEDS

48. PRE-SPROUTED
PRE SPROUTED SEED TEST
1. THREE YEARS OF TESTS
2. SELECT TREATMENTS GAVE GOOD
RESULTS
3. RATES WERE VERY IMPORTANT AS
HIGH RATES WERE PHYTOTOXIC
4. SAFE RATES WERE DETERMINED –
BUT ONLY THE VARIETY TRENASSE
WAS USED

49. TREATING PRE-SPROUTING
SEEDS
The Trenasse plots from foundation seed
(control, very light natural infection)
averaged 8254 lb/A at 12% moisture and
g
the inoculated check seed (diseased)
averaged 7366 lb/A and had a high level of
BPB. Pl
BPB Plots grown from seed pre-sprouted
f d d
in 0.1% acetic acid averaged 8631 lb/A, 3%
Starner = 8372 lb/A 7.5% Clorox = 8294
lb/A, 7 5%
lb/A, 1% copper sulfate = 8294 lb/A, and
0.6%
0 6% copper chloride = 8485 lb/A These
lb/A.
treatments had few panicles showing BPB.

50. a. Screening pesticides and timing of applications
b.
b Determining sources and genes for disease
resistance
c. Determining predisposing factors for disease
gp p g
development (effects of temperature on quorum
sensing and bacterial populations that cause
disease,
disease effects of nitrogen and other plant
nutrients on disease susceptibility and resistance,
effects of bacterial populations on seed and in soil
on disease development.
d. Determining genes associated with Quorum
sensing and attack mechanisms
e. Determining direct effects of temperature on
attack mechanisms
f. Determining temperature threshold that triggers
quorum sensing

51. CONCLUSIONS
Bacterial panicle blight is caused by
two bacterial pathogens that have been
p g
present on rice seeds and in soil (B.
gladioli) in rice producing areas of the
world for at least 60 years and probably
years,
much longer, usually causing minor
damage. Global warming has changed the
g g g
status of this disease to major status due to
the effects of increased temperatures on
quorum sensing BPB must now be
sensing.
researched vigorously to avoid severe
damage from epidemics as temperatures
g p p
continue to increase.

52. THANK YOU FOR
YOUR INTEREST
ARE THERE ANY
QUESTIONS?