Crown Gall Disease; Its Biology and Management Agrobacterium tumefaciens

Tom Burr

Cornell University, NYSAES

Geneva, NY

Rose Rose Raspberry

Cherry Aryganthemum

My Laboratory Works Primarily on Crown Gall of Grapevines, Caused by Agrobacterium vitis

• Agrobacterium vitis is specific to grape.

• Agrobacterium tumefaciens strains may also be

host specific however specific rose strains have

not been determined. In 1999 Pionnat et al.

reported a high level of genetic diversity among

A. tumefaciens strains from rose.

There are many similarities between crown gall on grapes and

crown gall on other plants such as rose.

Similarities Between Crown Gall of Different Plant Hosts

• A. vitis persists systemically throughout grapevine

• A. tumefaciens reported to be systemic in rose (M.

Lopez, 1999; demonstrated systemic colonization

and movement in rose. Also the pathogen was detected from

symptomless rose plants. Therefore it can be spread

in symptomless propagation material.

• Infections initiated at wounds; freeze injuries,

disbudding points, graft unions, etc.

• Plant injury is essential for crown gall initiation

on all plants.

Crown Gall in Vineyards and Nurseries

Grafts in nurseries

Lower trunks in

vineyard

*When young plants are infected they become stunted and

productivity is reduced.

Crown Gall at Grafts Inhibits Healing of the Graft Union

Union without Agrobacterium Union with Agrobacterium

Crown gall tissue; disorganized vascular development

T-DNA

Ti Plasmid

VirB pore nucleus

T-strand nuclear pore

VirD2

VirE2

VirE2

Lb Rb 3’

cell

wal

l

bac

teri

al m

embra

ne

VirE2

Attachment

VirA senses

Signals in

Plant wounds

VirG

VirG acitvates

other vir operons

Bacterium Plant Cytoplasm

Growth of A. vitis in graft union.

From about 500 cells to 1,000,000 within 5 days

2

3

4

5

6

7

Po

pu

lati

on

s (l

og

CF

U

0 5 18 30 60

Days after grafting

A. vitis Causes Necrosis on Grape

Grape necrosis

• Affects callus and vine

growth

• Inhibits graft take

• Provides niche for

survival of A. vitis in soil

• Do other Agrobacterium

cause necrosis on other

host plants?

Detecting A. vitis in grapevines using the MCH, Real-time PCR Assay

• MCH is more sensitive than previous methods for A. vitis. (at least 1000 times) • can detect about 10 cells of pathogen in a sample

• Is being used in the NCPN program to index accessions from foundation sources

• The gene target for real time PCR is sequence conserved in Agrobacterium virulence gene virD2. Also shown to amplify the virD2 product in A. tumefaciens strains from different hosts including rose. Can be used to test for presence of pathogen in host plants other than grape.

• Used to verify effectiveness of procedures for clean plant production

• To identify where pathogenic strains of Agrobacterium persists in the environment.

Vine number

Grapevine cane segment

1N 1I 2N 2I 3N 3I 4N 4I 5N 5I 6N 6I 7N 7I 8N 8I 9N 9I

1A - - - - - - - - - - - + + -

1B - - - - + + - - - - + -

2A - - + - + + - - - + - -

2B - - - - - + - - - - - -

3A + + + + - + - - - - - -

3B - - - - - + - - + - - -

4A - - + - - - - + - - - -

4B + + + - + + - + - + + + + +

5A + + - - + - - - + - - + + - + -

5B - - - - + - - - - - + - - - - - - -

6A - + + + - - - - - + - +

6B - + - + + - - + - -

7A - + + + - - - + + - + - + -

7B - - - - + - - - - +

8A - + + - - -

8B - - - - - - - - - - - - - -

9A + - - - - - - - + -

9B + - + + - + + + + -

10A - + - - - + + +

10B + - - - - + - -

Distribution of A. vitis in Canes

* Collected from vines with crown gall

Can A. vitis –free Grapevines be Produced and Maintained?

• Shoot tip and meristem culture as means to eliminate pathogens from plant material.

• How effective?

• What are environmental sources of pathogen that may contaminate the clean plants?

Can Clean Vines be Produced via Tissue Culture?

•2014-17 • Vines were propagated from tips and meristems (from a

crown gall-infected source, Riesling). • Tested by MCH method

• 5/18/16, green shoots from young vines • 5 of the 26 A. vitis tested positive

• 9/24/16, 0 of 26 tested positive

Can Clean Vines be Produced with Tissue Culture?

• 3/9/17, 6 nodes randomly selected from lignified shoots of each plant; 0 of 26 positive

• 3/9/17, 3 consecutive nodes from two lignified shoots from each vine. Collected 5 days after set above; 0 of 26 positive.

• 3/13/17, shoot tip and 6 consecutive green nodes and internodes for each vine. 0 of 26 positive.

Can Clean Vines be Produced with Tissue Culture?

• More vines have been initiated and are in greenhouse to be indexed.

• Conclusions: • Based on current results and most sensitive assay, plants “free” of A. vitis can

be generated. Only by more detailed evaluation like done in the 2017 experiments will we gain additional confidence.

• Can the bacterium persist at very low (less than 10 cells) or in a non-culturable form?

In addition to galls, A. vitis has been isolated from grapevine:

• Dormant grape buds • Range of 5 – 90% of buds positive for A. vitis.

• Shoot tips from vineyards with crown gall • Range of 7 – 30 % of shoot tips were positive for A. vitis

• Leaves from vineyards with crown gall • Range of 5 – 40% of leaves positive for A. vitis.

Conclusion: Pathogen can be detected in all parts of the vine; both internal and external. What factors trigger the initiation of infection? Injury and what others?

Wild Grapes as a source of A. vitis

• Wild grapes, NY – V. riparia • 30 of 95 (32%) vines positive for A. vitis

• Wild grapes, CA (feral seedlings and V. californica) • 25 of 87 (29%) positive for A. vitis

Sources of A. tumefaciens that would infect rose?

Biological Control of Crown Gall

The most studied biological control for crown gall is a

nonpathogenic strain of Agrobacterium isolated in Australia, strain

K84. It has been developed into a commercial product. Bacterial

cells are suspended in water and plants are soaked in the

suspension prior to planting.

Galltrol-A (AgBioChem, Inc.) = strain K84

Nogall (Bayer, Australia) = strain K1026 (engineered K84)

K84/K1026 does not control crown gall on all plants (grape)

Others being studied; F2/5, VAR03-1,

others.

Effectiveness of K84/K1026 for Crown Gall on Rose

• A few studies have been done but in total are inconclusive.

• Not all strains of A. tumefaciens are affected by K84. Most

notable examples of control have been on stone fruit crops.

• Study done by Farkas and Haas in 1985 concluded that K84

provided a high level of control of crown gall on rose.

• Research done by Marti et al in 1999 showed that of 84

isolates of A. tumefaciens from rose, only one was sensitive to

K84.

• Commentary from colleague in SA and from OR stated that

K84 has not been very effective for controlling crown gall of

rose.

Effectiveness of K84/K1026 for Crown Gall on Rose

• Agrobacterium radiobacter strain 84 has been used

successfully with roses in Australia, New Zealand, and

Spain but has not been effective in limited trials in the

United States. Strain K 84 is preventive only. Latent

infections (symptomless) and existing galls are not

controlled.

• One reason for differences of K84 effectiveness could

be related to the high level of diversity of A.

tumefaciens strains that have been shown to be

associated with rose.

Current Management Practices Cultivar/rootstock choice. Scion and rootstock varieties differ in susceptibility to crown gall.

Rose results. M. Lopez laboratory in Spain. 10 varieties all susceptible but gall size differed with variety (Sonia – Samantha). Best indication of differences could be gleaned from grower experience.

Sanitation. Once pathogen establishes systemically can be difficult to eradicate. Site selection. Plant on sites that have good air drainage and well drained soils to minimize freeze injury. Hilling up. Mounding soil over the graft union in the Fall protects it from extreme cold events, and ensures survival of scion buds for trunk renewal.

Current Management Practices

• Multiple trunks. Establishing multiple trunks allows for removal and replacement of galled trunks while maintaining production.

• Regular monitoring and replacement or renewal. Evaluate trunk and vine health on a regular basis, mark and replace trunks and vines.

• Cropping levels and fertility. Manage cropping levels, irrigation and nutrition such that active vegetative growth slows by veraison.

• Water management. Prevention of vigorous growth late in season

Acknowledgements

• USDA-APHIS NCPN

• USDA Federal Capacity Funds

• SCBG – USDA – New York State

• New York Wine and Grape Foundation

• Key Contributors

• Desen Zheng - Cherie Reid • Lingyun Hao - Tim Martinson • Kameka Johnson - Marc Fuchs