crown gall: current and future management methods
TRANSCRIPT
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Crown Gall: Current and Future Management Methods
Lani Yakabe Ali McCleanMalli Aradhya Jeff Moersfelder
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The crown gall pathogen
Agrobacterium tumefaciens
http://www.mindfully.org/GE/Vitaly-Citovsky-Projects.htm SCIMAT/SCIENCE PHOTO LIBRARYB2420004-Agrobacterium_tumefaciens-SPL.jpghttp://www.sciencephoto.com/media/13176/enlarge
- Gram negative soil-borne bacterium- free living or plant associated
Agro on aspen roots Agro on tobacco
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Walnut susceptibilitySome Juglans spp. are susceptible especially Paradox
‘Chandler’ in tissue culture NCB potted seedling J. hindsii potted seedling
orchard treesubterranean gallsfield seedlings
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Control of Crown Gall Disease• Fumigation/Chemical Control• Plant Clean Paradox Seeds• Implications of contaminated graft wood• “other approaches”
– K84 biocontrol– BioSI– Popcorn Sulfur
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Effi
cacy
–A
. tum
efac
iens A
A
B BBBB B
AA
B BBBB B
AA
B BBBB B
A
A
B BBBB B
A A
B BBBB B
AA
B BBBB B
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2’’-4’’ galls buried in sterile soil
Agro in Galls
A. tumefaciens Detected (120d)Treatment In galls In soilMeBr (400lb/acre ) 0/6* 0/6*TC35 (49 gal/acre ) 3/6 5/6TC35 + pic 0/8* 0/8*Non-fumigated control 16/16 14/16
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A. tumefaciens Recolonization
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0 10 20 30 40 50 60 70 80 90 100 110 120
days after reintroduction
log
CFU/
g so
il
MeBr Native C35 Sterile
AB
BC
A
B
C
A
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• MeBr and many alternatives control Agrobacterium tumefaciens
– 1,3-D only “reduces” A. tumefaciens populations– A combination of 1,3-D and chloropicrin (Telone C35)
best alternative for control of Agrobacterium tumefaciens/crown gall
• Fumigation alters the soil microbial community; result, pathogens can re-colonize to higher levels than in non-fumigated soil
Conclusions
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Long term soil survival
Determine A. tumefacienspopulations in soil pouches
Dig up pouch aftergiven time interval
Soil surface
18in
a) C35 trt soilb) Native soil
+A. tumefaciens
Two soil conditions: a.) fallowb.) orchard
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Soil Survival of A. tumefaciensOrchard Row
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1 2 3 4 5 6 7
Sample Times
A. t
umef
acie
ns
popu
latio
ns
C35 treated soil
Native Soil
1. Time 02. 7 days3. 70 days4. 110 days5. 210 days6. 270 days7. 365 days
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Control of Crown Gall Disease• Fumigation/Chemical Control• Plant Clean Paradox Seeds• Implications of contaminated graft wood• “other approaches”
– K84 biocontrol– BioSI– Popcorn Sulfur
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Scenario 2. Scenario 1.
Does Agrobacterium tumefacienscontaminate hybrid seed?
source of inoculum? …mother tree?…orchard floor?
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Mother Block SurveyDirectly off tree Off orchard floor
(1,3,7,14,28 days on soil)
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shell/ embryo
husk
exterior
No Agro Detected
No Agro Detected
-inhibitors
No Agro Detected
Directly off tree(>2000 nuts examined)
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Off orchard floor (1,3,7,14,28 days on
soil)
shell/ embryo
husk
exterior
No Agro detected…yet
Virulent/Avirulent
Agro. Detected
Virulent/Avirulent
Agro. Detected
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0
20
40
60
80
100
120
0 5 10 15 20 25 30
days
% tr
ees
with
Agr
o de
tect
ion
virulentavirulent
Contact with the orchard floor increases the probability of picking up A. tumefaciens.
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• A. tumefaciens not detected on/in seeds collected directly from mother trees
•A. tumefaciens contaminates seeds which hit the orchard floor
•
seed inoculum summary
exterior huskshell/
embryo
SO WHAT ??
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1) Biological vacuum generated by MeBr fumigated soil results in… no/limited microbial antagonists
MeBr fumigated soil
New direction: modifying soil-borne microbial communitiesin fumigated soils
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Control of Crown Gall Disease• Fumigation/Chemical Control• Plant Clean Paradox Seeds• Implications of contaminated graft wood• “other approaches”
– K84 biocontrol– BioSI– Popcorn Sulfur
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Determine source of inoculum and role of surface contaminated cuttings in crown gall development
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Is there a solution?
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Objectives
• Develop treatment for graft wood– heat : 55°C for 60 min
55°C for 45 min53°C for 60 min
– surfactant : BC/CTAB 5000 ppm for 20 min
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0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9
ppm
% R
educ
tion
of A
. tum
efac
iens
BC CTAB hypochlorite Physan
Surfactants – external contamination
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1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
20 25 30 35 40 45 50 55 60 65
time (min)
log
A. tu
mef
acie
ns (C
FU/m
l)
40C 45C 50C 55C 60C
Heat – external/internal contamination
104°F
113°F
122°F131°F140°F
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Control of Crown Gall Disease
• Fumigation/Chemical Control• Plant Clean Paradox Seeds• Implications of contaminated graft wood• “other approaches”
– K84 biocontrol (no dip tanks)– BioSI– Popcorn Sulfur
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Evaluation of wild Juglans species for crown gall resistance
Daniel Kluepfel1, Malli Aradhya1, Jeff Moersfelder1, Ali McClean1, Diane Velasco1,
and Wes Hackett2
1USDA-ARS, 2University of California Davis, CA
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Walnut Germplasm Screenseedlings in greenhouse conditions
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Walnut species screened previouslyJ. ailantifoliaJ. californicaJ. cathyensis
J. hindsiiJ. major
J. hopeinsisJ. mandshuricaJ. microcarpa
J. nigraJ. regia
J. sinensisPterocarya sp. (wingnut)
J. microcarpa and Pterocarya species exhibited the most crown gall resistance after multiple annual
screenings
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Discarded Plants
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Discarded Plants
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Resistant Plants
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Resistant Plants
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Progress in Genetic Mapping of Crown Gall Resistance
J. microcarpa (CG resistant) X J. regia ‘Serr’ (CG susceptible)A “new” Paradox-like rootstock
F1 hybrid progeny was propagated
54 seedlings were screened for CG resistance
Resistant individuals will be re-evaluated in the spring
Preliminary assessment: 35/54 with galls and 19/54 with no galls
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31.09A x ‘Serr’ hybrids
Preliminary results
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“Suggestions” for limiting Crown Gall
Limit exposure of paradox seeds & graft wood to soil.
Surface sterilize grafting tools frequently.
Limit time between nursery pickup and planting.
Fumigate planting hole/avoid replanting in same hole
Treat bare roots with Galltrol K84(?) (no dip tanks).
Limit wounding.
Avoid planting too deep.
Avoid mounding soil up on newly planted trees.
Keep crown of tree dry.
“The 9 point program”
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Crown Gall Update:
Lani Yakabe Ali McCleanMalli Aradhya Jeff Moersfelder