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Breeding to Mitigate HLB
in Citrus
Jude Grosser, Fred Gmitter, and Bill Castle
UF-CREC Citrus Genetic Improvement Team
2015
Scion Breeding
Jude Grosser, Fred Gmitter & Bill Castle
UF-CREC Citrus Genetic Improvement Team
2014
Plant species have thrived for thousands of years in the presence
of evolving, hostile pathogens – HOW? They have created their
own genetic diversity, and through the process of natural
selection, tolerant or resistant genotypes overcome the threat
and allow the species to evolve.
In Citrus, this process has been largely interrupted by man, with
Citriculture now approaching monoculture – leading to the
problem that has brought us all together.
Facilitated by biotechnology, citrus breeders have the
opportunity to artificially reinstate this process by creating
broad and unique genetic diversity from elite parents, followed
by robust screening. Maybe this is the answer for solving the
HLB and other disease problems!
Can we breed seedless (or low-seeded) sweet orange
hybrids tolerant of HLB? Maybe! Above tree is a 3x
hybrid containing 8% trifoliate orange, with juice
indistinguishable from commercial sweet oranges!
Midseason fruit with 37 color score and high brix.
Wide hybridization capturing HLB resistance
genes from trifoliate orange
2012
2013
2014
A Grand Experiment in Natural Selection: HLB-tolerant tetraploid breeding parent
Hirado Buntan Pink pummelo x 4x Succari sweet orange; PCR+ for HLB for
4 years, shows blotchy mottle in fall, but no effect on fruit!
Sugar Belle® is HLB tolerant! Trees near Fort Pierce,
HLB+ for more than six years – normal production!
Sugar Belle® is HLB tolerant! Young tree with HLB 3+
years, 4 boxes of beautiful fruit at Christmas
UF 7-6-27 ‘Bingo’ – easy-peel, seedless mandarin
approved for release – showing good HLB tolerance!
Cybrid 304: tree HLB+ more than 3 years. Large-fruited
mandarin; fruit not affected by HLB; juice has strong orange
note, amenable to processing!
Pummelos – an under-utilized genetic resource;
parent of sweet orange, grapefruit and rootstocks!
UF/CREC pummelo selections showing tolerance/resistance to HLB
and citrus canker (all are from open pollination; healthy trees in
high HLB/canker pressure grove).
Selection
5-1-99-3
Source
Hirado Buntan
pink
Fruit size
Large
Fruit shape
Globose
Peel thickness
Med
Flesh color
Pink
5-4-99-3
8-1-99-1B
Florida pummelo
Ling Ping Yau sdlg.
XXL
Large
Globose
Globose
Thin
Med
Pink
Pink
UKP-1
8-1-99-5B
Unknown
Ling Ping Yau sdlg.
Large
XL
Globose
Pyriform
Med
Med
Red
Red
HBJL-11
Hirado Buntan
pink
Florida pummelo
Siamese sweet
Hirado Buntan
pink
Florida pummelo
Ling Ping Yau sdlg.
XL
Globose
Med
Red
Large
Large
XL
Globose
Obovoid
Globose
Med/thin
Med
Thin
White
White
Red
XL
Large
Obovoid
Obovoid
Med
Med
White
White
5-4-99-7-S2
7-3-99-2
5-1-99-2-S5
5-4-99-1-S7
8-1-99-1A
When it comes to HLB, all
processing sweet orange clones
are not the same!
Differential response of sweet orange
clones to HLB
UF-CREC Citrus Genetic Improvement Team
2013
Sampling the OLL sweet orange somaclones.
OLL clones have superior quality and are showing
better stress tolerance than Valencia (including HLB)
ORANGES
‘OLL-8’
 Key attributes: Excellent color and quality, extends
harvest window of ‘Valencia’ quality juice
 Produces round oranges with internal and external
color similar to ‘Rhode Red Valencia’
 Holds on the tree exceptionally well, and maintains
quality into the summer
 Trees appear to yield better than standard ‘Valencia’
 High juice content and good pounds solids
 Peels easier than a standard ‘Valencia’
 With its added color, could also be a valuable addition
to the Florida fresh market portfolio
 Most precocious bearing clone among the OLL
somaclones
Juice Quality Data from OLL-Series Somaclones
Data 3-2-2012
Variety
Lbs. Juice
Per Box
Acid
Total
Brix
Ratio
Lbs. Solids
Per Box
Color
Valencia Control
OLL 20
OLL 27
OLL 25
OLL 9
OLL 15
OLL 2
OLL 3
OLL 4
OLL 7
Valencia control/SW
OLL 21
OLL 10
OLL 5
OLL 23
OLL 19
OLL 1
OLL 6
OLL 3-10 dwarf
OLL 8
54.41
53.23
54.99
57.58
56.30
49.48
51.55
54.52
55.52
57.51
48.46
58.20
55.55
53.92
57.21
54.02
55.10
58.19
53.80
56.43
1.00
0.84
0.84
0.79
0.94
0.99
0.77
0.98
0.92
0.94
0.79
0.77
0.82
0.93
0.81
0.84
0.90
0.96
0.80
0.77
12.03
12.10
12.34
12.81
12.97
14.56
12.77
12.92
12.60
13.20
12.91
12.38
12.42
13.13
12.12
12.32
12.90
12.85
12.51
11.99
12.03
14.40
14.69
16.22
13.80
14.71
16.58
13.18
13.70
14.04
16.34
16.08
15.15
14.12
14.96
14.67
14.33
13.39
15.64
15.57
6.55
6.44
6.79
7.38
7.30
7.20
6.58
7.04
7.00
7.59
6.26
7.20
6.90
7.08
6.93
6.65
7.11
7.48
6.73
6.77
38.60
40.50
39.80
40.20
39.90
39.50
41.00
40.30
40.70
41.20
39.10
40.30
40.60
40.10
39.90
40.90
41.50
40.80
41.20
40.70
‘OLL-8’
For more information on
‘OLL-8’ or ‘OLL-4’, please
contact Florida Foundation
Seed Producers, Inc.
www.ffsp.net
‘OLL-4’
‘OLL-4’
 Key attributes: excellent color and quality, extends
harvest window of ‘Valencia’ quality juice; also believed
to be higher yielding than ‘Valencia’
 Produces fruit with excellent internal and external
quality with exceptional juice color scores, juice content
and soluble solids
 Holds on the tree exceptionally well
 Maintains quality into the summer; however, it matured
earlier, and with better ratios than ‘Valencia’ in 2014
 Has been the highest yielding tree among the OLL
somaclones
Sweet orange OLL #7 topworked onto severely symptomatic
HLB-infected Valencia on Swingle. Tree treated with Harrell’s
UF mix CRF. Fruit set 2nd year.
Scion/Rootstock interaction – Synergy against HLB
- 3 trees Valencia N7-3/White grapefruit + 50-7
- only orange trees in block free of HLB symptoms
Breeding Rootstocks for the
HLB World
Jude Grosser, Fred Gmitter & Bill Castle
UF-CREC Citrus Genetic Improvement Team
2014
Differential Response to HLB
from rootstock candidates not
pre-selected for HLB tolerance
Citranges
Jude Grosser, Fred Gmitter, and Bill Castle
UF-CREC Citrus Genetic Improvement Team
2015
Premier Grapefruit Rootstock Trial – Fort Pierce
Premier Grapefruit Rootstock Trial – Fort Pierce
Several new citranges performing well!
HLB Severity and Yield
UFR-7,8,9,10,11&12 -best citranges
Differential Response to HLB
from rootstock candidates not
pre-selected for HLB tolerance
Sour Orange-like genetics
(half pummelo/half mandarin)
* Potential for wide soil adaptation and tolerance to blight!
Jude Grosser, Fred Gmitter, and Bill Castle
UF-CREC Citrus Genetic Improvement Team
2015
2013
2014
Valencia/HBPxCleopatra 46x20-04-48
4/4.5-year old resets in high HLB/blight pressure area
Ruby Red/HBPxCleopatra 46x20-04-S13
3-year old resets in high HLB pressure area (Vero)
Grapefruit on a allotetraploid hybrid of Amblycarpa + Pummelo – no HLB!
Premier Trial – Indian River (tetraploid sour orange-type rootstock)
Seed tree of allotetraploid hybrid of Amblycarpa + Pummelo – no HLB!
Several hybrid seed trees showing exceptional health.
St. Helena Trial 2012 4 years
Project c/o Mr. Orie Lee – Dundee Florida
Now more than 80 rootstocks under evaluation.
Mid-season Vernia and Valquarius scions
Trees grown with 100% CRF, evolving formulas.
Heavy HLB pressure (bad neighbor effect) entire time.
2014
6.5 years
NEW STRATEGY: BREEDING SOMATIC
HYBRID ROOTSTOCKS AT THE
TETRAPLOID LEVEL – CREATION OF
‘TETRAZYGS’
-Use of allotetraploid somatic hybrid breeding parents
allows the mixing of genes from 3-4 diploid rootstocks
at once.
- Progeny can be screened at the seed/seedling
level for wide soil adaptability and Phytophthora
resistance.
- Products can have direct rootstock potential including
adequate polylembryony, ability to control tree size due
to polyploidy, and improved disease resistance.
Candidate
for ACPS
Valquarius on Orange #15 tetrazyg rootstock – just < 5 years at
St. Helena, Dundee FL – released as UFR-3
Kuharske – 86% HLB frequency
Swingle – 70% HLB frequency
Orange #15 – 14% HLB frequency
Orange #19 –
23% HLB frequency
HLB-infected trees in the St. Helena Project
–differences in infection frequency & disease severity
Rootstock Data from 5-year old trees in the St. Helena trial - Dundee.
White = diploid; Orange = somatic hybrid; Blue = tetrazyg
NS = not significant fruit na = data not available * = control commercial rootstock Commercial control rootstocks in red
Lbs Solids/Box
Scion
Yield Boxes/Tree
Rootstock
2012
2013
2011 (35
mo.)
2012 (47
mo.)
2013 (59
mo.)
Cumulative Yield
(Boxes)
VALQUARIUS
UFR-6 CH+50-7
5.64
5.43
0.5
0.78
1.94
3.22
VERNIA
UFR-6 CH+50-7
5.67
6.01
0.4
0.63
1.41
2.44
VALQUARIUS
UFR-1 ORANGE 3
5.5
4.87
NS
0.72
2.23
2.95
VERNIA
UFR-1 ORANGE 3
5.61
6.28
0.31
0.67
1.33
2.31
VERNIA
UFR-2 ORANGE 4
5.47
5.93
0.35
0.25
1.38
1.98
VALQUARIUS
UFR-2 ORANGE 4
4.57
5.37
NS
0.75
1.73
2.48
VALQUARIUS
UFR-3 ORANGE 15
4.84
5.05
NS
0.81
1.97
2.78
VERNIA
UFR-3 ORANGE 15
5.46
5.82
0.37
0.38
1.82
2.57
VERNIA
UFR-4 ORANGE 19
5.79
6.07
0.54
0.71
1.73
2.98
VALQUARIUS
UFR-4 ORANGE 19
4.65
5.07
NS
0.65
1.59
2.64
VALQUARIUS
UFR-5 WHITE 4
5.76
5.72
0.33
0.56
1.80
2.69
VERNIA
UFR-5 WHITE 4
5.89
5.34
0.42
0.25
1.93
2.60
VALQUARIUS
UFR-13 FG 1731
5.83
6.81
NS
0.68
2.20
2.88
VALQUARIUS
UFR-14 FG 1733
5.12
5.63
NS
0.67
2.77
3.44
VERNIA
SWINGLE*
5.11
5.79
0.33
0.85
1.08
2.26
VALQUARIUS
SWINGLE*
NS
5.61
NS
NS
1.50
1.50
VERNIA
CLEO*
4.79
5.51
NS
0.50
0.83
1.33
VALQUARIUS
CLEO*
NS
5.21
NS
NS
1.7
1.7
VERNIA
R. LEMON*
3.67
na
NS
0.78
na
0.78
VALQUARIUS
VOLK*
NS
4.12
NS
NS
2.58
2.58
VERNIA
VOLK*
3.6
4.73
0.4
1.13
0.83
2.36
VALQUARIUS
KUHARSKE*
NS
5.75
NS
NS
2.2
2.2
VERNIA
KUHARSKE*
4.34
5.83
0.15
0.75
1.08
1.98
Tress With
Symptoms as of
March 2013
Number of
Trees in
Trial
Percentage with
HLB as of March,
2013
(5 years)
25
60
42%
15
60
25%
22
73
30%
6
43
14%
30
129
23%
20
72
28%
1
5
20%
14
20
70%
6
16
38%
12
18
67%
18
20
90%
56
65
86%
Table 3. Tree removal based on performance was initiated in the summer of 2014. Trees not expected to make a profit
during the 2015 harvest were removed for resetting. Tree removal data per diploid and tetraploid rootstocks provided
below. HLB was the cause of most of the removals (there were a few trees removed due to Phytophthora, and a few to wrap
around root systems). * indicates trees planted 18 months later (now 5 years old).
Rootstock
Diploid
UFR-16 46X31-02-13*
46X31-02-5*
68-1G-26-F4-P2
UFR-13 FG 1731
UFR-14 FG 1733
Orange 1804
Volk
Yellow 1800
68-1G-26-F6-P20
Rough Lemon
46X31-02-11*
68-1G-26-F2-P12
Swingle
46X31-02-S3*
Aqua 1803
White 1805
68-1G-26-F4-P6
HBJL-2B (n)*
48-OP-01
Cleo
Kuharske Carrizo
46X31-02-S9*
Pink 1802
MG11
White 1801
46X31-02-9*
Chandler A1-11*
Number of Trees in
Trial
Trees Removed 2014
Percent Trees Removed
due to Poor
Performance
8
13
12
5
5
18
20
11
17
18
15
10
20
19
19
19
13
23
28
16
63
15
18
40
11
18
8
0
0
0
0
0
0
0
0
1
1
1
1
2
2
2
2
2
4
5
3
12
3
4
12
4
3
3
0%
0%
0%
0%
0%
0%
0%
0%
6%
6%
7%
10%
10%
11%
11%
11%
15%
17%
18%
19%
19%
20%
22%
30%
36%
38%
38%
Table 3 (cont.) Tree removal based on performance was initiated in the summer of 2014. Trees not expected to make a profit
during the 2015 harvest were removed for resetting. Tree removal data per diploid and tetraploid rootstocks provided below.
HLB was the cause of most of the removals (there were a few trees removed due to Phytophthora, and a few to wrap around root
systems). * indicates trees planted 18 months later (now 5 years old).
Rootstock
Tetraploid
6058X6056-00-2*
A-MAC*
AMB+BENT*
SO+RPXSH 99-4*
SR+SH-99-11*
SR+SH-99-6*
UFR-17 Green 2*
UFR-3 Orange 15
WGFT+50-7
Blue 4
UFR-5 White 4
Purple 4
SO+CZO
Blue 9
Chang+Bent
UFR-1 Orange 3
Blue 1
Blue 2
Orange 13
Orange 18
UFR-2 Orange 4
White 1
UFR-4 Orange 19
AMB+5-1-99-2*
Orange 14
6058X2071-01-02*
Orange 16
SO+50-7
Orange 1
Number of Trees
19
19
20
26
7
6
16
43
86
37
72
64
265
30
34
60
69
24
50
45
70
24
128
18
62
27
27
45
24
Trees removed 2014
0
0
0
0
0
0
0
0
0
5
1
1
4
1
1
2
3
1
2
2
3
1
6
1
4
2
2
3
2
Percent Trees Removed
due to Poor Performance
0%
0%
0%
0%
0%
0%
0%
0%
0%
1%
1%
2%
2%
3%
3%
3%
4%
4%
4%
4%
4%
4%
5%
6%
6%
7%
7%
7%
8%
Susceptible
rootstock
Orange #1
Tolerant?
Rootstock
Green #7
Screening complex rootstock hybrids by growing Valencia scion from HLB-infected
budwood. Left 3 trees: rootstock Orange # 1 (Nova+HBP x Cleo+trifoliate orange);
Right 3 trees: rootstock Green #7 (Nova+HBPummelo x Sour orange+Carrizo)
Research article: Rootstock-regulated gene expression patterns
associated with fire blight resistance in apple
By Philip J Jensen1, Noemi Halbrendt1,2, Gennaro Fazio3, Izabela Makalowska4,
Naomi Altman5, Craig Praul6, Siela N Maximova7, Henry K Ngugi1,2, Robert M
Crassweller7, James W Travis1,2 and Timothy W McNellis1*
BMC Genomics 2012, 13:9
In apple, rootstock genetics effect scion gene expression – in this case affecting
fire blight resistance (also caused by a gram-negative bacterium). Thus,
something being produced by the rootstock is being translocated to the scion that
affects disease resistance – why wouldn’t this happen in citrus as well, especially
with complex tetraploid rootstocks?
Rootstocks differentially translocate nutrients, phytohormones (plant growth
regulators), micro-RNAs, small proteins (pathogenesis related?), and other
metabolites to the scion. This could have both direct and indirect, quantitative
and quantitative affects on scion gene expression, and possibly Lilberibacter
pathogenesis in citrus – especially with unique complex allotetraploid rootstocks.
The New Gauntlet in the HLB world
1.
2.
3.
4.
5.
6.
7.
Crosses of superior parents made at diploid and tetraploid levels
Seed harvested from crosses planted in bins of calcareous soil (pH=8),
inoculated with P. nicotianae and P. palmivora (JH Graham)
Selection of robust seedlings based on growth rate, health and color
(most don’t make it!)
Transfer to 4x4 pots in commercial potting soil
Top of new tree goes for seed source tree production; remaining liner
to the HLB screen
Hybrid liner is grafted with HLB-infected budstick of Valencia sweet
orange; remaining rootstock top removed, forced flushing from HLBinfected sweet orange budstick
Trees monitored for HLB symptoms – healthy appearing trees entered
into ‘hot psyllid’ house for 4 weeks, followed by field planting at Picos
Farm (under DPI permit).
Seed from diploid and tetraploid crosses are planted in
calcareous soil inoculated with P. nicotianae and P. palmivora
(Diane Bright/Graham lab). This cross shows high susceptibility
to Phytophthora.
Rootstock cross with good Phytophthora resistance.
Gauntlet trees are produced by ‘stick’ grafts. HLB-infected
Valencia budsticks wrapped in parafilm are grafted into selected
rootstock candidates. Rootstock tops are used to produce rooted
cuttings for seed trees on their own roots.
Flush from HLB-infected budsticks grows right through
the parafilm, no unwrapping is necessary.
Quite often the first flush is symptom free, selection is
based on the 2nd flush, which usually shows symptoms.
Initially Susceptible (including dead trees) – The Duds
HLB screening of complex new rootstock candidates by grafting ‘hot’ PCR+ HLB-infected
Valencia budsticks into each hybrid (after propagation of seed trees). Valencia trees
growing out from the infected tissue with little or no symptoms after the 2nd flush are
passed through a ‘hot’ psyllid house prior to field planting at the UDSA Picos Farm.
Intermediate response – these are saved for observation;
on rare occasions a few show a recovery phenomenon.
Good First Flush
Good Second Flush
Candidates on right are passed through the ‘hot psyllid’ house until feeding
damage is observed, then trees planted at USDA-Picos Farm.
Gauntlet Survivor at Picos Farm
-Valencia on A+Volk x Orange #19-11-31
Gauntlet Survivor at Picos Farm
-Valencia on Milam+HBP x Orange #14-09-14
Gauntlet Survivor at Picos Farm
-Valencia on A+HBJL-2 x Orange #19-08-2
HLB Tolerance or Resistance/Avoidance?
60 trees of Valencia somaclones on Orange #19 rootstock (UFR-4),
graft-inoculated twice in the Dawson HLB greenhouse were planted
at the USDA-Picos Farm in Fort Pierce, Spring of 2011.
Orange #19: Nova+HBPummelo x Cleo+Argentine trifoliate orange –
a tetrazyg produced from conventional breeding of somatic hybrids.
57 trees are still in this trial (3 removed due to poor health), 9 tested
PCR-negative (15.8%) in December, 2014. Does this suggest a partial
resistance or avoidance mechanism in the rootstock? If so, can it be
enhanced by further breeding?
Juice Quality Data from Two New Early-Maturing Valencia Somaclones.
Data from fruit harvested first week of December, 2014, 6-year old trees
on rough lemon rootstock, Alligator Grove, St. Cloud, Florida.
Lbs.
Juice Acid
Per
Box
Total
Brix
Fruit
Ct
Lbs.
Lbs.
Solids Solids juice
Per Box Per Box color
53.906
0.87
11.04
12.69
78
5.9512
5.95
35.3
B7-70
50.913
0.71
11.30
15.92
61
5.7532
5.75
36
Hamlin
52.914
0.94
11.17
11.88
64
5.9105
5.91
34.5
Valuarius
50.728
0.84
9.87
11.75
55
5.0069
5.01
35.7
SF14W-65 50.299
0.67
11.06
16.51
63
5.5631
5.56
36
Valencia
0.98
9.53
9.72
58
5.0931
5.09
35.4
Variety
Vernia
53.443
Ratio
PCR-negativeValencia/Orange #19 (UFR-4) rootstock, inoculated twice
in Dawson HLB greenhouse, now at USDA-Picos Farm for nearly 4 years!
PCR testing of Gauntlet Trees at Picos Farm – December, 2014.
PCR-negative trees with excellent health and vigor (January, 2015):
1. A+Volk x Orange 19-11-9
4x
2. A+FD x Orange 19-11-10
4x
3. B21-R5-T25-11-10
2x (Flying Dragon Bull)
PCR-positive trees with excellent health and vigor (January, 2015):
1. A+HBJL-2B x Orange 19-09-9 4x
2. Milam+HBP x Orange 14-9-6 4x
3. A+HBJL-2B-OP-9-36
4x
A = Amblycarpa
FD = Flying Dragon
HBJL-2B = pummelo selection
OP = open pollination
Rootstock improvements regarding HLB are like
likely to come in stages:
First stage: Rootstocks that reduce the frequency of HLB
infection, and reduce the severity of the disease once infected –
these will still require efficient psyllid control and optimized
production systems.
Second stage: Potential rootstock mitigation of the disease –
research is underway to possibly identify rootstocks that can
protect the entire tree – regardless of the scion. Psyllid control
may not be necessary. No horticultural performance data would
be available on such selections initially, but the hybrids would
have good rootstock pedigree, and can be mass-propagated by
tissue culture (Ruck’s Nursery, Agristarts, Agromillora, etc.).
To HALL OF FAME CITRUS GROWER-RESEARCHER
and Outstanding Industry Collaborators: Mr. Orie Lee
Funding: Mr. Orie Lee, Citrus Variety Improvement Grants from the Citrus
Research and Development Foundation (CRDF) , USDA/CSREES; and the
Citrus Research and Education Foundation (CREF).
Thanks also to: Cecile Robertson (Dawson lab), Mike Irey and the SG
Diagnostic Lab, Diane Bright (Graham lab), Angel Hoyte and Lukasz
Stelinski (hot psyllid house), Gary Barthe, Chuck Dunning, Mauricio Rubio,
Ralph ‘Chandler’ Story, Ed Stover and Steve Mao & the USDA Picos Farm
Crew, many others, and especially Troy Gainey and the CREC Grove Crew
(including our scouts!).
Thanks!
UF-CREC Citrus Genetic Improvement Team
2015