Crown Gall Disease; Its Biology and Management ... · Biological Control of Crown Gall The most...
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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