BACTERIAL PANICLE BLIGHT:
   CAUSES AND SUGGESTED
    CONTROLL MEASURES

Milton C. Rush, Donald E. Groth, Jong Ham,
      ...
Rice
   Ri produced i th southern
             d d in the       th
United States has a long history of
loss to panicle bli...
Panicle blighting had been attributed to
           g    g
 abiotic factors including high
 temperatures, water stress, or...
PATHOGENS
• FURTHER STUDIES INDICATED THAT TWO
  PLANT PATHOGENIC BACTERIA CAUSED THE
  EPIDEMICS OF PANICLE BLIGHTING

• ...
Bacterial Panicle Blight
Inoculated     Non inoculated
               Non-inoculated
SEVERE BPB
More than 400 isolates of the two pathogens
were isolated from diseased plants collected across
the southern United States...
Burkholderia glumae
PCR analysis of DNA isolated from B glumae (top) and B gladioli
                                         B.               ...
Bacterial Panicle Blight in Panama


   We also observed the disease on rice
in Panama in 2002 and 2005. I
received sample...
The disease was later reported from other
Asian countries and Latin America. B. glumae
p
produces the p y
               p...
This di
    Thi disease is characterized as
                   i h     t i d
having upright, straw-colored panicles
contai...
Bacterial Panicle Blight
BACTERIAL PANICLE BLIGHT




 SPRAY INOCULATED – 106 CFU / ML (quorum sensing)
Effect of Temperature on Growth of
           BPB pathogens
   Effect of temperature on the growth of
B. glumae (A) and B....
EFFECT OF TEMPERATURE ON
GROWTH OF Burkholderia glumae
and Burkholderia gladioli STRAINS
                            1 .8 ...
The temperature optima
ranged b t
       d between 38 and 40°C
                        d
(
(100-104F) for B. glumae and
  ...
Bacterial Panicle Blight in the
         U.S. in 2010
TEMPERATURES were in the range of
90-100F (32-38C) during the day an...
NIGHT TEMPERATURES
   From our observations, it
             observations
appears that extended high
 pp                  ...
Quorum Sensing
   This is a type of decision-making
                     decision making
process used by decentralized gro...
A variety of diff
         i t f different
                       t
molecules can be used as
signals. A common class of
si...
Activation of the receptor induces
the up regulation of other specific g
     p g                      p     genes,
      ...
Virulence Factors Produced By Burkholderia
                             y
      glumae in Response to Quorum Sensing

• Kn...
Temperature and Quorum Sensing: Relationship
            to BPB Disease Development
      It is known that the production ...
Effect of loss of quorum Sensing Signals
   in Avirulent Strains of B. glumae
    Virulence of the B. glumae strains was
c...
Testing for Virulence




Onion scale inoculation   Inoculation of rice
Flagella Production




Virulent         Avirulent
Toxoflavin Toxin Production




  No
  N toxoflavin
        fl i     Toxoflavin
                 T fl i (yellow)
Inoculating Entries in the BPB Nursery
Three Row Plot in the BPB Nursery
      Center Row Inoculated
Susceptible Breeding Line
S     tibl B di Li
LM-1: Resistance Source for BPB Developed by
   D.E.
   D E Groth using Cobalt Irradiation of Var
                        ...
Susceptible Commercial Var.
            Cocodrie
Crossing to Transfer Resistance
         g

 Crosses with Resistant Entries
           LM –1, AB 647, LR 2065
           N...
SOURCES OF RESISTANCE TO BPB
1.
1 JUPITER – U S MEDIUM GRAIN VARIETY
            U.S.             VARIETY,
   STUDIES UNDE...
Effect of B. glumae on the Yield Potential of Rice
          B




      Difference between sprayed and unsprayed p
      ...
Effects of Panicle Blight on Selected
Commercial Varieties - 2005
              Yield (lb/A at 12% Moisture)     Differenc...
Effects of Soilborne B. gladioli
   on Yield of Bengal Rice
Soil strains sprayed                      Yield (lb/A at
     ...
Chemical Control of BPB
   Oxolinic acid (Starner) is the only
effective chemical control available
for this pathogen when...
QUARANTINE
   In this context use of pathogen free
           context,        pathogen-free
seeds is an important practice...
Testing Seeds

•    We d l d th d f t ti
     W developed methods for testing
    seed with PCR and Real-Time PCR
       a...
CONTROLLING BPB
1. OXOLINIC ACID/STARNER (JAPAN)
2. COPPER FUNGICIDES ????
3.
3 SEED TREATMENT
4. TREATMENT OF SEED IN PRE...
HEAT TREATMENT OF RICE
 SEEDS TO CONTROL BPB

• TREATMENT OF DRY SEED FOR 5-6 DAYS
  AT 65°C

• WET TREATMENT OF SEEDS AT ...
TREATING PRE-SPROUTED
SEEDS
                        3

     1




 2                      4
Stand counts
Trial II -
Beginning of Heading
Diseased Panicle




  INFECTION FROM INFECTED SEEDS
PRE-SPROUTED
  PRE SPROUTED SEED TEST
1. THREE YEARS OF TESTS
2. SELECT TREATMENTS GAVE GOOD
   RESULTS
3. RATES WERE VERY...
TREATING PRE-SPROUTING
        SEEDS
The Trenasse plots from foundation seed
(control, very light natural infection)
avera...
a. Screening pesticides and timing of applications
b.
b Determining sources and genes for disease
   resistance
c. Determi...
CONCLUSIONS
    Bacterial panicle blight is caused by
two bacterial pathogens that have been
                p    g
presen...
THANK YOU FOR
 YOUR INTEREST

ARE THERE ANY
  QUESTIONS?
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001 bacterial panicle blight, milton rush

  1. 1. BACTERIAL PANICLE BLIGHT: CAUSES AND SUGGESTED CONTROLL MEASURES Milton C. Rush, Donald E. Groth, Jong Ham, And R. Nandakumar Louisiana State University
  2. 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. 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. 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. 5. Bacterial Panicle Blight Inoculated Non inoculated Non-inoculated
  6. 6. SEVERE BPB
  7. 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. 8. Burkholderia glumae
  9. 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. 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. 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. 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. 13. Bacterial Panicle Blight
  14. 14. BACTERIAL PANICLE BLIGHT SPRAY INOCULATED – 106 CFU / ML (quorum sensing)
  15. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 26. Testing for Virulence Onion scale inoculation Inoculation of rice
  27. 27. Flagella Production Virulent Avirulent
  28. 28. Toxoflavin Toxin Production No N toxoflavin fl i Toxoflavin T fl i (yellow)
  29. 29. Inoculating Entries in the BPB Nursery
  30. 30. Three Row Plot in the BPB Nursery Center Row Inoculated
  31. 31. Susceptible Breeding Line S tibl B di Li
  32. 32. LM-1: Resistance Source for BPB Developed by D.E. D E Groth using Cobalt Irradiation of Var Var. Lemont
  33. 33. Susceptible Commercial Var. Cocodrie
  34. 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. 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. 36. Effect of B. glumae on the Yield Potential of Rice B Difference between sprayed and unsprayed p p y p y plots
  37. 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. 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. 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. 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. 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. 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. 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. 44. TREATING PRE-SPROUTED SEEDS 3 1 2 4
  45. 45. Stand counts
  46. 46. Trial II - Beginning of Heading
  47. 47. Diseased Panicle INFECTION FROM INFECTED SEEDS
  48. 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. 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. 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. 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. 52. THANK YOU FOR YOUR INTEREST ARE THERE ANY QUESTIONS?

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