Using Stigma Imprints to Determine
Fire Blight Risk and Improve Control of
Blossom Blight
Scott C. Ockey
Utah State Horticulture Association annual meeting
Provo, Utah
January 26-28, 2004
Stigma Imprint Background:
• Fire blight bacteria multiply on the
stigmatic surfaces of blossoms.
• Stigma imprints indicate presence of
bacteria on the stigma surfaces and
percentage of blossoms within an
orchard with epiphytic bacterial
populations.
• Stigma imprint results can be used to
guide bactericide applications.
• Current stigma imprinting protocols
provide results in 36-48 hrs.
2003 Objectives:
• Improve timeliness of stigma imprinting
results by bringing stigma imprinting
closer to home.
• Improve timeliness of stigma imprinting by
implementing current molecular detection
procedures.
• Continue to work on stigma imprinting
technique and evaluate stigma imprint
data.
Improve timeliness of stigma
imprinting results.
• We continued having Lester perform
stigma sampling.
– Previously poured Petri plates were on
hand at Mountainland.
– Lester followed an established sampling
protocol in each selected orchard.
• We brought the lab to Mountainland.
– Incubator—reduced results to 24-30 hours.
– Microscope—Verified ease of identification.
Molecular Detection Procedure
rtPCRreal time Polymerase Chain Reaction
•Used a 944bp segment of DNA common
to all virulent Fire Blight Bacteria.
•Modified PCR protocol published by Al
Jones et al. to rtPCR.
•Used Idaho Technologies R.A.P.I.D.
rtPCR unit.
•Set up molecular grade lab at
Mountainland packing shed.
What Happened?????
• Strong inhibitors present on stigmatic and
blossom surfaces.
• Length of replicated DNA strand to long for
rtPCR procedure.
• Complex of both factors.
What Next???
• Evaluate shorter DNA sequences.
– 447bp sequence found PEANT.
– New protocol tested.
• Work with published protocols for
removing inhibitors.
– Use of Llop extraction procedure developed
specifically for bacterial plant pathogens.
Testing done on
crabapple stigma
and blossom
using PEANT
primers and/or
Llop extraction
protocol.
Continue to work on stigma imprinting
technique and evaluate stigma imprint data.
•Stigma imprinting with Streptomycin
amended media provides resistance
information.
No Strep
Strep
2003 Stigma Imprint Data for Santaquin #2 Gala
20.00
70.00
18.00
60.00
50.00
14.00
12.00
40.00
10.00
9
30.00
8.00
6.00
20.00
4.00
3
10.00
2.00
0
0
0
0
0
1
1
1
1
1
1
1
1
1
0
0
0
0
Sample Date
05/19/03
05/18/03
05/17/03
05/16/03
05/15/03
05/14/03
05/13/03
05/12/03
05/11/03
05/10/03
05/09/03
05/08/03
05/07/03
05/06/03
05/05/03
05/04/03
05/03/03
05/02/03
05/01/03
04/30/03
04/29/03
04/28/03
04/27/03
04/26/03
04/25/03
0.00
04/24/03
0.00
•Fire blight Bacteria present before Maryblyt, Cougarblight or the
temerature mean models predicted.
•Stigma imprints indicated that 62% of the colonized
flowers had streptomycin resistant strains.
•Suggested spraying blossoms with Mycoshield.
24-hr Mean Temperature
% Positive Stigma Imprints
16.00
2003 Stigma Imprint Data for Pear block #2
20.00
70.00
18.00
60.00
14.00
13
12
12.00
11
50.00
40.00
10.00
30.00
8.00
7
6.00
4.00
20.00
24-hr Mean Temperature
% Positive Stigma Imprints
16.00
4
10.00
2.00
0
05/02/03
05/01/03
04/30/03
04/29/03
04/28/03
04/27/03
04/26/03
04/25/03
04/24/03
04/23/03
0.00
04/22/03
0.00
Sample Date
•Fire blight Bacteria present before Maryblyt, Cougarblight or the
temerature mean models predicted.
•Stigma imprints indicated that most colonized
flowers had streptomycin resistant strains.
•No sprays applied, 8strikes/tree some trees near cankers had 95%
blossoms infected. Infection occurred at low temps.
•Blossom blight infections can and do occur
when climatic conditions and models
suggest otherwise.
•Numerous over wintering cankers
increases blossom blight risk even with
non-conducive climatic conditions.
•Two row planting systems inhibit spray
movement, resulting in spray coverage
problems.
•Streptomycin and Mycoshield applications
do not reduce bacterial populations on the
stigma. However, they do coat the floral
cup to reduce infection potential.
Stigma imprints may be able to help
identify “hot spots” within an orchard.
•Set a sampling route to be followed each
sampling day.
•Mark plates to correspond to points on the
sampling route.
•Check for bacterial growth on plates and
determine if there is an occurrence pattern.
•Go back to the orchard and check for
overlooked cankers or other causes
(beehives).
We would like to thank:
USHA
Mountainland
Grower Cooperators
Idaho Technologies
Doug Rowley
Lester
Ryan Black