BACTERIAL PANICLE BLIGHT:BACTERIAL PANICLE BLIGHT: CAUSES AND SUGGESTED

CONTROLL MEASURESCONTROLL MEASURES

Milton C. Rush, Donald E. Groth, Jong Ham,And R. NandakumarAnd R. Nandakumar

Louisiana State University

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

Panicle blighting had been attributed to g gabiotic factors including high temperatures, water stress, or toxic pchemicals near the root zone, but in 1996-97 the bacterial plant pathogen p p gBurkholderia 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 grain rotting p g gand seedling blighting in 1956.

PATHOGENSPATHOGENS• FURTHER STUDIES INDICATED THAT TWO

PLANT PATHOGENIC BACTERIA CAUSED THE EPIDEMICS OF PANICLE BLIGHTING

• Burkholderia glumaeSEEDBORNE– SEEDBORNE

• Burkholderia gladiolig– SEEDBORNE– SOILBORNE

Bacterial Panicle BlightInoculated Non inoculatedInoculated Non-inoculated

SEVERE BPBSEVERE BPB

More than 400 isolates of the two pathogens were isolated from diseased plants collected acrosswere 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 yanalysis, and PCR. The B. glumae pathogen was determined to be the same AS an ATTC isolate of h h f J hi h fi dthe pathogen from Japan, which was first reported

in 1956 as causing grain and seedling rot on rice.

Burkholderia glumae

PCR analysis of DNA isolated from B glumae (top) and B gladioliPCR analysis of DNA isolated from B. glumae (top) and B. gladioli (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 (ATCCLane 1, 100 bp ladder; lane 2, positive control (top) B. glumae (ATCC 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 11 121 2 3 4 5 6 7 8 9 10 11 12

Bacterial Panicle Blight in PanamaBacterial Panicle Blight in Panama

We also observed the disease on rice in Panama in 2002 and 2005. I received samples in 2006 from which pwe isolated both B. glumae and B. gladioli based on PCR and othergladioli based on PCR and other identification procedures.

The disease was later reported from otherThe disease was later reported from other Asian countries and Latin America. B. glumaeproduces the phytotoxin, toxoflavin, which is p p y , ,essential for its virulence and strains that lack toxin production usually become avirulent.

Th t f b t i l i l bli htThe symptoms of bacterial panicle blight include seedling blight, sheath rot lesions on the flag-leaf sheath, and panicle blighting withflag leaf sheath, and panicle blighting with significant yield losses. Specific leaf sheath symptoms include vertical lesions with gray

t d d b d k ddi h bcenters surrounded by a dark reddish brown margin.

Thi di i h t i dThis disease is characterized as having upright, straw-colored panicles containing florets with a darker basecontaining florets with a darker base and a reddish-brown line (margin of lesion) across the floret between the d k d th t l ddarker area and the straw-colored area, resulting in abortion of the kernel before it fills. The severely affected ypanicles remain upright, as the grain does not fill. The term “panicle blight” has been used in the United States forhas 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.

Bacterial Panicle BlightBacterial Panicle Blight

BACTERIAL PANICLE BLIGHT

SPRAY INOCULATED 106 CFU / ML (quorum sensing)SPRAY INOCULATED – 106 CFU / ML (quorum sensing)

Effect of Temperature on Growth of BPB pathogens

Effect of temperature on the growth ofEffect of temperature on the growth of B. glumae (A) and B. gladioli (B) strains. Each line represents growth of a single p g gstrain after 48 h of incubation in KBB. Each strain was tested with three replicates. Ten

i d f h i (b dstrains were tested for each species (based on PCR and fatty acid identification). Strains were isolated from rice panicleStrains were isolated from rice panicle collections showing symptoms of bacterial panicle blight from Louisiana Texas andpanicle blight from Louisiana, Texas, and Arkansas.

EFFECT OF TEMPERATURE ON GROWTH OF Burkholderia glumaeGROWTH OF Burkholderia glumae and Burkholderia gladioli STRAINS

1 .4

1 .6

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1 .8

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nsity

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00 n

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0 .8

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ical

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3 0 3 5 4 0 4 5 5 00

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3 0 3 5 4 0 4 5T e m p e r a t u r e C T e m p e r a t u r e C

Burkholderia glumae strains Burkholderia gladioli strains

The temperature optima d b t 38 d 40°Cranged between 38 and 40°C

(100-104F) for B. glumae and ( ) g35 and 37°C (95-99F) for B.

l di li Th ltgladioli. These results were confirmed with repeatedconfirmed with repeated experiments.

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 ythe 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 gboth states according to Extension Specialists with yield losses to 50% in p ysome fields.

NIGHT TEMPERATURESNIGHT TEMPERATURES

From our observations itFrom our observations, it appears that extended high pp gtemperatures at night seem to b i t t f t i BPBbe an important factor in BPB disease development.p

Quorum SensingQuorum Sensing

This is a type of decision makingThis is a type of decision-making process used by decentralized groups to coordinate behavior. Many species of bacterial pathogens use quorumof bacterial pathogens use quorum sensing to coordinate their gene

i di t th l lexpression according to the local density of their population. y p p

A i t f diff tA variety of different molecules can be used as signals. A common class of i li l l i Gsignaling molecules in Gram-

negative bacteria, such as B.negative bacteria, such as B. glumae and B. gladioli, is N-Acyl Homoserine Lactones (AHL)(AHL)

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

Virulence Factors Produced By Burkholderia yglumae in Response to Quorum Sensing

• Known: toxoflavin toxin, lipase, flagella formation (QsmR), catalase (KatG)(Q ), ( )

• Possible factors: type III secretion system,t ll l l h idextracellular polysaccharides

Temperature and Quorum Sensing: Relationship to BPB Disease Developmentto BPB Disease Development

It is known that the production of the major virulence factors of B glumae; toxoflavin lipasevirulence factors of B. glumae; toxoflavin, lipase and flagella, are dependent on the quorum-sensing system mediated by AHL signal

S i imolecules. Several virulent and avirulent B. glumae strains were tested for their ability to produce AHL signal molecules using an AHL-produce AHL signal molecules using an AHLbiosensor strain, Chromobacterium violaceum CV026. Interestingly, all the strains that produce

f th i l f t t t d d f tinone of the virulence factors tested were defective in AHL-signal biosynthesis, while the strains that produce at least one of the major virulence p jfactors showed AHL-positive phenotypes.

Effect of loss of quorum Sensing SignalsEffect of loss of quorum Sensing Signals in Avirulent Strains of B. glumaeVirulence of the B. glumae strains was

closely related to their ability to produce various virulence factors Interestingly allvarious virulence factors. Interestingly, all 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 insensing signals implying that mutation in global regulatory system(s) for the virulence factors is the major cause of the occurrence

f i l i iof avirulent B. glumae strains in nature

Testing for VirulenceTesting for Virulence

Onion scale inoculation Inoculation of riceOnion scale inoculation Inoculation of rice

Flagella ProductionFlagella Production

Virulent AvirulentAvirulent

Toxoflavin Toxin ProductionToxoflavin Toxin Production

N fl i T fl iNo toxoflavin Toxoflavin (yellow)

Inoculating Entries in the BPB NurseryInoculating Entries in the BPB Nursery

Three Row Plot in the BPB Nursery Center Row Inoculated

S tibl B di LiSusceptible Breeding Line

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

Lemont

Susceptible Commercial Var. Cocodrie

Crossing to Transfer Resistanceg

Crosses with Resistant EntriesCrosses with Resistant EntriesLM –1, AB 647, LR 2065Nipponbare JupiterNipponbare, Jupiter

Crosses were made with the susceptible varieties

CCDR, CPRS, and FRNS to study inheritanceCCDR, CPRS, and FRNS to study inheritance

of the resistance in these materials.

Results

SOURCES OF RESISTANCE TO BPBSOURCES OF RESISTANCE TO BPB

1 JUPITER – U S MEDIUM GRAIN VARIETY1. JUPITER U.S. MEDIUM GRAIN 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

Effect of B glumae on the Yield Potential of RiceEffect of B. glumae on the Yield Potential of Rice

Difference between sprayed and unsprayed plotsp y p y p

Effects of Panicle Blight on SelectedEffects of Panicle Blight on Selected Commercial Varieties - 2005

VarietiesYield (lb/A at 12% Moisture) Difference

(lb/A at 12% moisture)Non-

i l t dInoculated

inoculatedYield Rating

Yield Rating

Cocodrie 8047 7001 1047nsCocodrie 8047 0.8

7001 7.3

- 1047ns

Jupiter 10,168 0 5

9731 3 3

- 437ns0.5 3.3

Trenasse 8338 2.3

6687 8.3

- 1651**

Bengal 8260 6262 1978**Bengal 8260 2.7

6262 8.7

- 1978**

Effects of Soilborne B. gladiolion Yield of Bengal Rice

Soil strains sprayedRating

Yield (lb/A at 12 % Moist re)Rating 12 % Moisture)

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 (S il B l di li) 3 7 88543S5 (Soil B. gladioli) 3.7 8854 c

S15 (Soil B. gladioli) 4.3 8822 bc

ATCC B. gladioli 4.3 8609 bcATCC B. gladioli 4.3 8609 bc

336gr-1 B. glumae 8.7 6282 a

Chemical Control of BPBChemical Control of BPB

Oxolinic acid (Starner) is the onlyOxolinic 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 andisolated from rice in Japan and oxolinic acid is not labeled for use on rice in the United Statesrice in the United States.

QUARANTINEQUARANTINE

In this context use of pathogen freeIn this context, use of pathogen-free seeds is an important practice to

d th i id freduce or manage the incidence of BPB. Therefore, it was essential to develop rapid, sensitive and inexpensive methods for identifying p y gand quantifying the levels of B. glumaein certified seeds.in certified seeds.

Testing SeedsTesting Seeds

W d l d th d f t ti• We developed methods for testing seed with PCR and Real-Time PCR

a. Indirect methodb Direct methodb. Direct method

• Semi-selective media (S-Pg and CCNT)CCNT)

CONTROLLING BPBCONTROLLING BPB1. OXOLINIC ACID/STARNER (JAPAN)1. OXOLINIC ACID/STARNER (JAPAN)2. COPPER FUNGICIDES ????3 SEED TREATMENT3. SEED TREATMENT4. TREATMENT OF SEED IN PRE-

SPROUTING WATER5. HEAT TREATMENT OF SEED6. DISEASE RESISTANCE7 QUARANTINE7. 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 DRYDRY

TREATING PRE-SPROUTED SEEDS

1

3

2 4

Stand counts

Trial II -Beginning of HeadingBeginning of Heading

Diseased Panicle

INFECTION FROM INFECTED SEEDS

PRE SPROUTED SEED TESTPRE-SPROUTED SEED TEST1. THREE YEARS OF TESTS1. THREE YEARS OF TESTS2. SELECT TREATMENTS GAVE GOOD

RESULTSRESULTS3. RATES WERE VERY IMPORTANT AS

HIGH RATES WERE PHYTOTOXICHIGH RATES WERE PHYTOTOXIC4. SAFE RATES WERE DETERMINED –

BUT ONLY THE VARIETY TRENASSEBUT ONLY THE VARIETY TRENASSE WAS USED

TREATING PRE-SPROUTING SEEDSSEEDS

The Trenasse plots from foundation seedThe 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 of BPB Pl f d dBPB. Plots grown from seed pre-sprouted in 0.1% acetic acid averaged 8631 lb/A, 3% Starner = 8372 lb/A 7 5% Clorox = 8294Starner = 8372 lb/A, 7.5% Clorox = 8294 lb/A, 1% copper sulfate = 8294 lb/A, and 0 6% copper chloride = 8485 lb/A These0.6% copper chloride 8485 lb/A. These treatments had few panicles showing BPB.

a. Screening pesticides and timing of applicationsb Determining sources and genes for diseaseb. Determining sources and genes for disease

resistancec. Determining predisposing factors for disease g p p g

development (effects of temperature on quorum sensing and bacterial populations that cause disease effects of nitrogen and other plantdisease, 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 mechanismssensing and attack mechanisms

e. Determining direct effects of temperature on attack mechanismsattack mechanisms

f. Determining temperature threshold that triggers quorum sensing

CONCLUSIONSBacterial panicle blight is caused by

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

THANK YOU FOR YOUR INTEREST

ARE THERE ANYARE THERE ANY QUESTIONS?QUESTIONS?