How to Live with HLB

How to Live with HLB?
Yongping Duan and Melissa Doud
USDA ARS USHRL
Fort Pierce, FL
‘Candidatus Liberibacter asiaticus’
Diaphorna citri
•Phloem-limited
•Systemic infection with a
long latent period(6 mo-3 years)
Elimination or Suppression of
HLB Bacteria by:
Thermotherapy
 Chemotherapy
Hypothesis:
1) Heat and /or chemical treatments (“stress”) will
induce a conversion from Las prophage to phage
(from lysogenic to lytic cycles), and possibly a
conversion from a “severe strain” to a “mild
strain”.
2) High temperature (40-45oC) will impose stress
conditions both on Las bacterium directly and from
host to Las bacteria indirectly.
Heat treatment (40oC) added beneficial effects on HLBaffected trees
Heat + chemical
treatments
Chemical
treatment only
Chemical Induction of a Conversion from a “Severe
Strain” to a “Mild Strain”
Blotchy Mottle
Treated with
Non-effective
chemical
Treated with
Nanosilver
Treated with
SAR
Temperature
40°C
42°C (19h) 30°C (5h)
45°C (16h) 30°C (8h)
No Treatment Control
A
B
Time
(days)
4
4
4
270
Initial
30 DATa
DAT
Ct value Ct value
Ct value
19.34
21.68
ND
18.42
20.56
ND
22.47
N/A
ND
21.57
N/A
28.78
A
B
HLB-affected citrus (A) Before heat treatment and (B) 6 months after exposure to heat
Will thermotherapy mitigate
HLB in the field?
Major factors affect the effectiveness
 Fluctuating temperatures
 Soil temperatures
 Humidity
 Large scale with cost effectiveness
Tents
A
B
Portable greenhouses for thermotherapy (A) Clear tents - allow
100% of light through which can result in tissue damage; (B)
Opaque tents - allow 80% of light which decreases leaf burn to a
minimum, keeps humidity closer to 100%, and plastic is 11 mil
Gro-Tec™ woven polyethylene. Both tents are made by
“Flowerhouse”.
Thermotherapy Dooryard Citrus
PROOF OF CONCEPT
•Dooryard Sour Orange Citrus experiment began August 2011
•24 trees were trimmed and placed in portable greenhouses for 7
days
•Tented trees exposed to 33-55 hours above 104°C
• 6 trees were trimmed and not tented
•13-4-9 fertilizer applied after treatment and in March 2012
Las bacteria initially decreases but will return
10000000
Cells per PCR sample
1000000
100000
10000
Heated 1
Heated 2
Heated 3
Heated 4
Control
1000
100
10
1
Before
6 months
12 months
18 months
Below line
is
considered
PCR
negative
HLB Dooryard Citrus – No Treatment
Initial
9 months later
*No foliar nutrition, no pesticides, subpar amounts of time release fertilizer
1.5 years later
Treated HLB Dooryard Citrus Increases in Growth
Initial
9 months later
1.5 years later
2.5 years later
Growth maintained for 1.5 years but then declines possibly due to poor management e.g.
no foliar nutrition, no pesticides, subpar amounts of time release fertilizer
Subpar response to field thermotherapy
Before
1.5 years later
*No foliar nutrition, no pesticides, subpar amounts of time release fertilizer
2.5 years later
Concept Proven
• Trees exposed to thermal therapy have less Las after heat treatment for two years
BUT titer increases over time
• Trees not exposed to heat remain Las positive with similar bacterial levels or
increasing levels
• Trees produce more flush after treatment for 2-3 yrs when managed properly
BUT THERE ARE PROBLEMS
Heat treatment is not a cure, Las is in roots possibly trunk
Las not killed will spread again through the tree and reinfection by psyllid is possible
Declining HLB trees usually see an increase in flush and growth after heat treatment
but NOT ALL trees respond as well, WHY?
Treated trees may begin to show sparse canopies but WHEN?
Picos Farm Therapy July 2012 – September 2013
•HLB Valencia on Carrizo rootstock planted summer 2009
•Tented throughout the year for 3-14 days
Tissue Damage is Possible
A
B
•
•
•
•
3 yr old Valencia on Carrizo
Tented for 7 days in mid July 2012 with few rain storms
>74 hours above 104°F, 135°F highest, 24 hrs above 122°F (A)
Reduce length of treatment and/or vent tent (B)
August 2012 – 3 Day Treatment
122°F
100% RH
113°F
104°F
2 PM
2 PM
2 PM
2 PM
Mid October 2012– 13 Day Treatment
Before
10 months after treatment
17 months after treatment
Mid October-13 days of Treatment
Hours
150
100
50
0
50-59
60-69
70-79
80-89
90-99
Fahrenheit
100-109
110-119
120-129
July 2013– 7 Day Treatment and 9 months later
August 2013– 4 Day Treatment and 6 months later
Edsall Commercial Grove Spring 2011
• Homemade tent using visqueen, PVC pipes, and
cement blocks
Edsall Commercial Grove Spring 2011
Strong positive response to heat treatment
Before
May 2011
20 months later
Jan 2013
3 years later
April 2014
Edsall HLB trees almost 3 years after heat treatment – April 2014
Edsall Commercial Grove Spring 2011
Not all trees respond as strongly
Before
May 2011
2 years later
2013
3 years later
April 2014
Edsall Commercial Grove Spring 2012
Before
May 2012
8 months later
Jan 2013
2 years later
April 2014
Edsall Commercial Grove Spring 2012
Before
May 2012
8 months later
Jan 2013
2 years later
April 2014
Does thermal therapy affect fruit drop?
2 years after
treatment
April 2014
No
treatment
Fruit Production Field Trial
• Three field locations
• 4-year-old and 15-year-old Valencia on various
rootstocks
• Randomized Block Design
• Trees tented for >72 hours (includes 3
afternoons of sunshine) during late AugustSeptember 2013
• Most 4-yr-old trees responded with large
flushes
• Initial response on 15-yr-old trees was not as
drastic
• Changes in fruit production will be recorded
from this season and compared to next season
(2015)
Initial Results
from Fall 2013
Trial
Before August 2013
After
March 2014
Thermotherapy at Commercial Grove
• Draped 6 mil visqueen over 4 15-yr old Valencia trees
• Held down by soil
• 100 ft roll of visqueen can cover 8 of these trees
Before
August 2013
After
April 2014
• 15-year-old Valencia with high titer
• Draped visqueen caused tissue damage
• Only regions of increased flush
Heat Stress Causes Changes in Citrus
• Heat stress is known to alter functions of RNA and proteins in
plants
• Proteomics and RNA-Seq both identify heat-induced changes in
citrus
• Can we manipulate these changes using a chemical or another
stressor?
Summary
• HLB-affected citrus exposed to high temperatures respond
favorably (maximize high heat duration while minimizing tissue
damage)
• Trees grow more vigorously while HLB bacterial levels decrease
for a period of time
• Improvement in tree growth has been observed for 3 years
• Severely sick trees may NOT respond well to the heat treatment
• Field trials to track changes in fruit production are ongoing
• Currently we are working on improving the treatment and on
revealing the molecular mechanism of heat treatment on HLB
bacteria and citrus
Acknowledgements
Duan Lab Previous and Current:
•
Dr. Melissa Doud
• Dr. Michele Hoffman
• Christina Latza
• Meredith Hickman
• Taylor Hurt
• Andrea Folds
• Wei Chieh Lee
• Greg Brock
•
•
•
Dr. MQ Zhang, UF
Dr. C.A. Powell, UF
Dr. Ed Stover, USDA
• Greenhouse and Picos
staff
• Guettler family
• Edsall grove
• Blue Goose grove
• Evans properties
Funding provided by
USDA-ARS, FDACS
SCBG Grant and
CRDF grant
Plastics/Temperatures – Field Discussion
• 11 mil Gro-Tec™ woven polyethylene
• Visqueen
• Top of tents hotter than at the bottom
• Bigger trees can withstand hotter temperatures for longer durations
• USDA often treats trees in the summer for 72 hours (must include 3
afternoons of sunny skies with only quick storms) so that the tent in
the middle reaches above 104°F for 2-6 hours each day
• Late October to early March is usually too cool for thermotherapy
and an additional heat source is recommended
• Late September and early October treatments initially respond well
but subsequent growth is not as vigorous as summer treatments
After Treatments – Field Discussion
• Expect some tissue damage at the top of the trees
• Leaves that existed before heat treatment will still show HLB
symptoms after heat treatment
• New flush within a month will appear
• Flush is a major attractant for pysllids and leaf miner
• Not all trees respond as well to treatment
• 3-4 and 7-8 yr old trees showed typical response but 15 yr old trees
showed smaller flushes
• Trees without management e.g. trimming, pesticides, and nutrition
showed decline within 2-2.5 yrs after heat treatment