Crown Gall: Current and Future Management Methods

Crown Gall: Current and Future Management Methods

Lani Yakabe Ali McCleanMalli Aradhya Jeff Moersfelder

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

Walnut susceptibilitySome Juglans spp. are susceptible especially Paradox

‘Chandler’ in tissue culture NCB potted seedling J. hindsii potted seedling

orchard treesubterranean gallsfield seedlings

Control of Crown Gall Disease• Fumigation/Chemical Control• Plant Clean Paradox Seeds• Implications of contaminated graft wood• “other approaches”

– K84 biocontrol– BioSI– Popcorn Sulfur

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

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

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

• 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

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

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

Scenario 2. Scenario 1.

Does Agrobacterium tumefacienscontaminate hybrid seed?

source of inoculum? …mother tree?…orchard floor?

Mother Block SurveyDirectly off tree Off orchard floor

(1,3,7,14,28 days on soil)

shell/ embryo

husk

exterior

No Agro Detected

No Agro Detected

-inhibitors

No Agro Detected

Directly off tree(>2000 nuts examined)

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

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.

• 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 ??

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

Determine source of inoculum and role of surface contaminated cuttings in crown gall development

Is there a solution?

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

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

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

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

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

Walnut Germplasm Screenseedlings in greenhouse conditions

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

Discarded Plants

Resistant Plants

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

31.09A x ‘Serr’ hybrids

Preliminary results

“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”

Crown Gall Update:

Lani Yakabe Ali McCleanMalli Aradhya Jeff Moersfelder

Crown Gall: Current and Future Management Methods

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