Crown Gall: Current and Future Management Methods

Crown Gall: Current and Future Management Methods Lani Yakabe Ali McClean Malli Aradhya Jeff Moersfelder The crown gall pathogen Agrobacterium tum...
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Crown Gall: Current and Future Management Methods

Lani Yakabe Ali McClean Malli Aradhya Jeff Moersfelder

The crown gall pathogen

Agrobacterium tumefaciens Agro on aspen roots

http://www.mindfully.org/GE/Vitaly-Citovsky-Projects.htm

- Gram negative soil-borne bacterium - free living or plant associated

Agro on tobacco

SCIMAT/SCIENCE PHOTO LIBRARY B2420004-Agrobacterium_tumefaciens-SPL.jpg http://www.sciencephoto.com/media/13176/enlarge

Walnut susceptibility Some Juglans spp. are susceptible especially Paradox

‘Chandler’ in tissue culture

field seedlings

NCB potted seedling

subterranean galls

J. hindsii potted seedling

orchard tree

Control of Crown Gall Disease • • • •

Fumigation/Chemical Control Plant Clean Paradox Seeds Implications of contaminated graft wood “other approaches” – K84 biocontrol – BioSI – Popcorn Sulfur

Efficacy – A. tumefaciens

A

AA

AA

A

B BB

B BB

B B B

BB B

B B

B

B B B

A

AA

A A

A

B BB

B BB

B B B

B BB

B BB

B BB

Agro in Galls 

2’’-4’’ galls buried in sterile soil A. tumefaciens Detected (120d)

Treatment MeBr (400lb/acre ) TC35 (49 gal/acre ) TC35 + pic Non-fumigated control

In galls 0/6* 3/6 0/8* 16/16

In soil 0/6* 5/6 0/8* 14/16

lo g C F U /g so il

A. tumefaciens Recolonization 1.00E+07

A

1.00E+06

B

A

B

1.00E+05

C

A

1.00E+04

B

1.00E+03 1.00E+02

C

1.00E+01 1.00E+00 0

10

20

30

40

50

60

70

80

90

days after reintroduction MeBr

Native

C35

Sterile

100

110

120

Conclusions • 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

Long term soil survival a) C35 trt soil b) Native soil

Soil surface

+

18in

A. tumefaciens

Dig up pouch after given time interval

Two soil conditions: a.) fallow b.) orchard

Determine A. tumefaciens populations in soil pouches

Soil Survival of A. tumefaciens Orchard Row A. tumefaciens populations

1.00E+07 1.00E+06 1.00E+05

C35 treated soil

1.00E+04

Native Soil

1.00E+03 1.00E+02 1

2

3

4

5

Sample Times

6

7

1. 2. 3. 4. 5. 6. 7.

Time 0 7 days 70 days 110 days 210 days 270 days 365 days

Control of Crown Gall Disease • • • •

Fumigation/Chemical Control Plant Clean Paradox Seeds Implications of contaminated graft wood “other approaches” – K84 biocontrol – BioSI – Popcorn Sulfur

Does Agrobacterium tumefaciens contaminate hybrid seed? source of inoculum? …mother tree? …orchard floor?

Scenario 1.

Scenario 2.

Directly off tree

Off orchard floor (1,3,7,14,28 days on soil)

Mother Block Survey

No Agro Detected shell/ embryo

Directly off tree (>2000 nuts examined)

No Agro Detected -inhibitors husk

No Agro Detected exterior

No Agro detected…yet shell/ embryo

Virulent/Avirulent Agro. Detected husk

Virulent/Avirulent Agro. Detected exterior

Off orchard floor (1,3,7,14,28 days on soil)

120

% trees with Agro detection

100

80 virulent

60

avirulent

40

20

0 0

5

10

15

20

25

30

days

Contact with the orchard floor increases the probability of picking up A. tumefaciens.

seed inoculum summary • A. tumefaciens not detected on/in seeds collected directly from mother trees •A. tumefaciens contaminates seeds which hit the orchard floor



exterior

husk

shell/ embryo

SO WHAT ??

MeBr fumigated soil 1) Biological vacuum generated by MeBr fumigated soil results in… no/limited microbial antagonists

New direction: modifying soil-borne microbial communities in fumigated soils

Control of Crown Gall Disease • • • •

Fumigation/Chemical Control Plant Clean Paradox Seeds Implications of contaminated graft wood “other approaches” – K84 biocontrol – BioSI – Popcorn Sulfur

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 min 53°C for 60 min – surfactant : BC/CTAB 5000 ppm for 20 min

Surfactants – external contamination

% Reduction of A. tumefaciens

120 100 80 60 40 20 0 0

1

2

3

4

5

6

7

ppm BC

CTAB

hypochlorite

Physan

8

9

Heat – external/internal contamination 1.E+06

104°F log A. tum efaciens (CFU/ml)

1.E+05

113°F 1.E+04

1.E+03

1.E+02

122°F 131°F 140°F

1.E+01

1.E+00 20

25

30

35

40

45

50

55

time (min) 40C

45C

50C

55C

60C

60

65

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 Screen seedlings in greenhouse conditions

Walnut species screened previously J. ailantifolia J. californica J. cathyensis J. hindsii J. major J. hopeinsis J. mandshurica J. microcarpa J. nigra J. regia J. sinensis Pterocarya sp. (wingnut)

J. microcarpa and Pterocarya species exhibited the most crown gall resistance after multiple annual screenings

Discarded Plants

Discarded Plants

Resistant 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

Preliminary assessment: 35/54 with galls and 19/54 with no galls

Resistant individuals will be re-evaluated in the spring

31.09A x ‘Serr’ hybrids

Preliminary results

“Suggestions” for limiting Crown Gall “The 9 point program” 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.

Crown Gall Update:

Lani Yakabe Ali McClean Malli Aradhya Jeff Moersfelder