BACTERIAL PANICLE BLIGHT: CAUSES AND SUGGESTED CONTROLL MEASURESMilton C. Rush, Donald E. Groth, Jong Ham, And R. Nandakumar Louisiana State University
Rice Ri produced i th southern d d in the thUnited States has a long history ofloss to panicle blighting of unknownetiology. Epidemics of panicle blightoccurred during 1995 and 1998, yearsof record high temperatures, withyyield losses in some fields estimated tobe as high as 40%. Significant losseswere also experienced in Louisianaduringd i 2000 and 2010 b th years of d 2010, both funusually high temperature.
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.
PATHOGENS• FURTHER STUDIES INDICATED THAT TWO PLANT PATHOGENIC BACTERIA CAUSED THE EPIDEMICS OF PANICLE BLIGHTING• Burkholderia glumae – SEEDBORNE• Burkholderia gladioli g – SEEDBORNE – SOILBORNE
Bacterial Panicle BlightInoculated Non inoculated Non-inoculated
More than 400 isolates of the two pathogenswere isolated from diseased plants collected acrossthe southern United States rice area. The two pathogens were identified by growthon selective media, BiologTM, Cellular fatty acid g yanalysis, and PCR. The B. glumae pathogen wasdetermined to be the same AS an ATTC isolate ofthe pathogen f h h from JJapan, which was fi reported hi h first din 1956 as causing grain and seedling rot on rice.
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
Bacterial Panicle Blight in Panama We also observed the disease on ricein Panama in 2002 and 2005. Ireceived samples in 2006 from which pwe isolated both B. glumae and B.gladioli based on PCR and otheridentification procedures.
The disease was later reported from otherAsian countries and Latin America. B. glumaepproduces the p y phytotoxin, toxoflavin, which is , ,essential for its virulence and strains that lacktoxin production usually become avirulent. The Th symptoms of b t i l panicle blight t f bacterial i l bli htinclude seedling blight, sheath rot lesions on theflag leafflag-leaf sheath, and panicle blighting withsignificant yield losses. Specific leaf sheathsymptoms include vertical lesions with graycenters surrounded b a d k reddish b t d d by dark ddi h brownmargin.
This di Thi disease is characterized as i h t i dhaving upright, straw-colored paniclescontaining florets with a darker baseand a reddish-brown line (margin oflesion) across the floret between thedarkerd k area and th straw-colored area, d the t l dresulting in abortion of the kernelbefore it fills. The severely affected ypanicles remain upright, as the graindoes not fill. The term “panicle blight”has been used in the United States formore than 50 years and BPB has beenretained as the name for the disease inthis country.
Effect of Temperature on Growth of BPB pathogens Effect of temperature on the growth ofB. glumae (A) and B. gladioli (B) strains.Each line represents g p growth of a single gstrain after 48 h of incubation in KBB. Eachstrain was tested with three replicates. Tenstrains were tested for each species (b d i df h i (basedon PCR and fatty acid identification).Strains were isolated from rice paniclecollections showing symptoms of bacterialpanicle blight from Louisiana, Texas, and Louisiana TexasArkansas.
EFFECT OF TEMPERATURE ONGROWTH OF Burkholderia glumaeand Burkholderia gladioli STRAINS 1 .8 1 .8 1 .6 1 .6 1 .4 1 .4 nsity at 600 nm 1 .2 1 .2 1 1Optical 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 CBurkholderia glumae strains Burkholderia gladioli strains
The temperature optimaranged b t d between 38 and 40°C d((100-104F) for B. glumae and ) g35 and 37°C (95-99F) for B.gladioli. Th l di li These results were ltconfirmed with repeatedexperiments.
Bacterial Panicle Blight in the U.S. in 2010TEMPERATURES were in the range of90-100F (32-38C) during the day and in ( ) g ythe 80s (27-29C) at night in Louisianaand Arkansas in 2010. These were newrecords for night temperature (37states). Bacterial blighting was severe in ) g gboth states according to ExtensionSpecialists with y p yield losses to 50% insome fields.
NIGHT TEMPERATURES From our observations, it observationsappears that extended high pp gtemperatures at night seem tobeb an iimportant factor in BPB t tf t idisease development. p
Quorum Sensing This is a type of decision-making decision makingprocess used by decentralized groupsto coordinate behavior. Many speciesof bacterial pathogens use quorumsensing to coordinate their geneexpression according to the local i di t th l ldensity of their p p y population.
A variety of diff i t f different tmolecules can be used assignals. A common class ofsignaling molecules in Gram- i li l l i Gnegative bacteria, such as B.glumae and B. gladioli, isN-Acyl Homoserine Lactones(AHL)
Activation of the receptor inducesthe up regulation of other specific g p g p genes, ,causing all of the cells to begintranscription at approximately the same p pp ytime. This includes the turning on of theppathogen attack g g genes. Optimum ptemperatures for growth means that thebacterial population reaches the p pthreshold population to turn on theseggenes more q quickly. y
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
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.
Effect of loss of quorum Sensing Signals in Avirulent Strains of B. glumae Virulence of the B. glumae strains wasclosely related to their ability to producevarious virulence factors Interestingly all factors. Interestingly,the confirmed avirulent strains weredefective in multiple virulence factors andmost of them lost their ability to produceacyl-homoserine lactone (AHL) quorum-sensing signals implying that mutation inglobal regulatory system(s) for the virulencefactors is the major cause of the occurrenceof avirulent B. glumae strains in nature f i l i i
Testing for VirulenceOnion scale inoculation Inoculation of rice
Crossing to Transfer Resistance g Crosses with Resistant Entries LM –1, AB 647, LR 2065 Nipponbare, Nipponbare JupiterCrosses were made with the susceptible varietiesCCDR, CPRS, and FRNS to study inheritanceof the resistance in these materials. Results
SOURCES OF RESISTANCE TO BPB1.1 JUPITER – U S MEDIUM GRAIN VARIETY U.S. VARIETY, STUDIES UNDERWAY BY DR. JONG HAM AND HIS GRADUATE STUDENTS, , LOUISIANA STATE UNIVERSITY2. NIPPONBARRE, Teqing, LR 2065, LM-1, US HYBRED VARIETIES, OTHER MATERIALS
Effect of B. glumae on the Yield Potential of Rice B Difference between sprayed and unsprayed p p y p y plots
Effects of Panicle Blight on SelectedCommercial 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
Effects of Soilborne B. gladioli on Yield of Bengal RiceSoil strains sprayed Yield (lb/A at Rating 12 % Moisture) Moist re)Non-inoculated (Healthy control) 2.7 8260 bcS-10 (Soil B. gladioli) 3.7 7666 b223 gr-1 (Grain B. gladioli) 3.3 8659 bc3S4 (Soil B. gladioli) 2.7 8788 bc3S5 (Soil B gladioli) (S il B. l di li) 37 3.7 8854 cS15 (Soil B. gladioli) 4.3 8822 bcATCC B. gladioli 4.3 8609 bc336gr-1 B. glumae 8.7 6282 a
Chemical Control of BPB Oxolinic acid (Starner) is the onlyeffective chemical control availablefor this pathogen when used as aspray, however, oxolinic acid-resistant B. glumae strains have beenisolated from rice in Japan andoxolinic acid is not labeled for use onrice in the United States States.
QUARANTINE In this context use of pathogen free context, pathogen-freeseeds is an important practice toreduce or manage th i id d the incidence of fBPB. Therefore, it was essential todevelop rapid, sensitive andinexpensive methods for identifying p y gand quantifying the levels of B. glumaein certified seeds.
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)
CONTROLLING BPB1. OXOLINIC ACID/STARNER (JAPAN)2. COPPER FUNGICIDES ????3.3 SEED TREATMENT4. TREATMENT OF SEED IN PRE- SPROUTING WATER5. HEAT TREATMENT OF SEED6. DISEASE RESISTANCE7.7 QUARANTINE
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
Diseased Panicle INFECTION FROM INFECTED SEEDS
PRE-SPROUTED PRE SPROUTED SEED TEST1. THREE YEARS OF TESTS2. SELECT TREATMENTS GAVE GOOD RESULTS3. RATES WERE VERY IMPORTANT AS HIGH RATES WERE PHYTOTOXIC4. SAFE RATES WERE DETERMINED – BUT ONLY THE VARIETY TRENASSE WAS USED
TREATING PRE-SPROUTING SEEDSThe Trenasse plots from foundation seed(control, very light natural infection)averaged 8254 lb/A at 12% moisture and gthe inoculated check seed (diseased)averaged 7366 lb/A and had a high level ofBPB. PlBPB Plots grown from seed pre-sprouted f d din 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, and0.6%0 6% copper chloride = 8485 lb/A These lb/A.treatments had few panicles showing BPB.
a. Screening pesticides and timing of applicationsb.b Determining sources and genes for disease resistancec. 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 mechanismse. Determining direct effects of temperature on attack mechanismsf. Determining temperature threshold that triggers quorum sensing
CONCLUSIONS Bacterial panicle blight is caused bytwo bacterial pathogens that have been p gpresent on rice seeds and in soil (B.gladioli) in rice producing areas of theworld for at least 60 years and probably years,much longer, usually causing minordamage. Global warming has changed the g g gstatus of this disease to major status due tothe effects of increased temperatures onquorum sensing BPB must now be sensing.researched vigorously to avoid severedamage from epidemics as temperatures g p pcontinue to increase.
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