Bacteriophage can reduce Ralstonia solanacearum · PDF fileBacteriophage can reduce Ralstonia solanacearum induced stem ... AP2011 - Document not peer ... Phage stock solutions were

Bacteriophage can reduce Ralstonia solanacearum induced stem
necrosis in tobacco
Bruce Fortnum and Paul Peterson
CORESTA 2011
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Ralstonia solanaceasrum is a gram negative, non spore forming rod shaped bacterium that exhibits strong tissue-
specific tropism within the host invading primarily the xylem tissue.
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Mechanization is a major method of
Pathogen Spread following abrasion of stem tissue
Topping
Harvesting
Stalk destruction
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F 0 0 0 0 0 0 0 0 0 0 0 0 0 X X 0 0 0 0 0 0 0 0 0 0 0 0 0 B
Model
Machine Topped
F X X 0 X X X 0 0 0 X 0 0 X O X X X 0 X 0 X X X X 0 X X X B F 0 0 X 0 0 0 0 0 0 0 0 0 0 O X X X X X X X X X X X X X X B F 0 0 X X 0 0 0 0 X 0 X 0 X X X X X 0 0 X X X 0 0 0 X 0 0 B F X X 0 0 X X 0 0 X X 0 0 X O X X 0 0 X X X X 0 X X 0 X 0 B F X 0 X 0 0 0 X 0 0 0 0 X X X X X X X X X 0 X 0 0 0 X 0 0 B F 0 X X X X X X X X X X X X X X X X X X X X X X X 0 0 0 0 B F 0 X X 0 X X X X 0 X 0 X X X X X X X X X X 0 X X 0 0 0 0 B F 0 0 X X X X 0 0 X X X X X X X X 0 0 0 0 X X 0 0 X 0 0 0 B
At the end of the season 100% of plants are infected with R. solanacearum
0 = non-infected, X = infected with R. Solanacearum
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Bacteriaphage are natural components within many soil ecosystems
Breeding nurseries planted in monoculture with high populations of R. solanacearum decline in disease intensity over time
Disease loss surveys within South Carolina suggests some level of biocontrol is occuring in production fields
Bacteriaphage diverse group of biological entities
ssRNA, dsRNA, ssDNA, dsDNA
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Objective 1 Determine the incidence of R.
solanacearum Phage in South Carolina
tobacco fields
2 Evaluate efficacy of Phage to reduce stem
colonization in greenhouse trials
3 Field evaluation of Phage across R.
solanacearum strains
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Field Survey
Soil solution
Experimental design
Filter to remove bacteria (0.45 micron)
Tube containing 4.5 ml soft agar + 0.49 ml Y3 inoculum (107) + 100 ml Phage suspect material
Phage assay using the double agar method
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Sample number Presence of Phage Designation
PD 1 + F RSDILY
PD 2 + F RSPD
1 - ---
2 - ---
3 + F RS3
4 + F RS4
5 - ---
6 + F RS6
7 + F RS7
8 + F RS8
9 + F RS9
10 + F RS10
75% of samples were positive for R. solanacearum isolate Y3 Phage
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Experimental Design
Greenhouse Trials
RCB design - Treatments were replicated 4 times
The R. solanacearum culture (Y3) were grown on nutrient agar (72 hr), re-suspended in deionized water at OD600=0.2=10
8 cells / ml and used as a stock culture. The 108 suspension was used to make inoculum for a 107 dilution.
Phage stock solutions were filter sterilized (0.45 mm)
R. solanacearum (Y3) 4.5 ml + 500 ml Phage suspension were incubated for 48 h at 26 C.
100 ml of Y3+ phage suspension were applied to a cut tobacco stem and evaluated for disease over a 20 day period
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R. Solancearum Y3 - 982 bp
Greenhouse Trial
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Treatments Field Trial 1. SC10 Tobacco isolate 982 bp AVRa 2. NC 132 Tobacco isolate 982 bp AVRa 3. SC 06 Tobacco isolate - 982 bp AVRa 4. Y3 - Tobacco isolate - 982 bp AVRa 5. F RSDILY Phage isolate
Experimental Design Field Trials
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Field Trial 2009
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Field Trial 2010
R. Solancearum SC 10. SC 06, Y3 - 982 bp
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Treatment Stem Necrosis AUDPC
SC 10 4.7 a 50.6 a
NC 132 4.4 a 45.4 a
SC 10 + DILY 2.7 b 14.6 bc
NC 132 + DILY 2.8 b 21.4 b
DILY 0.2 c 0 d
UTC 0.1 c 0 d
Field Trial 2009
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Treatment Stem Necrosis AUDPC
SC 10 4.5 ab 51.4 a
SC 06 4.6 a 53.5 a
Y 3 4.2 ab 45.8 a
SC 10 + DILY 3.75 bc 42.1 a
SC 06 + DILY 2.5 d 12.6 cd
Y 3 + DILY 3.1 cd 26 b
DILY 0 f 0 d
UTC 0 f 0 d
Field Trial 2010 2011
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Summary R. solanacearum Phage are frequently found
in fields where disease is observed
Phage isolates reduced disease progression in Greenhouse and Field trials
Some Phage isolates can enhance R. solanacearum virulence
Data in agreement of work performed by Yamada et al. and Fujiwara et al.
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Documents

Bacteriophage can reduce Ralstonia solanacearum · PDF fileBacteriophage can reduce Ralstonia solanacearum induced stem ... AP2011 - Document not peer ... Phage stock solutions were