Breeding to Mitigate HLB in Citrus

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 Source Fruit size Fruit shape Peel thickness Flesh color 5-1-99-3 Hirado Buntan Large Globose Med Pink pink 5-4-99-3 Florida pummelo XXL Globose Thin Pink 8-1-99-1B Ling Ping Yau sdlg. Large Globose Med Pink UKP-1 Unknown Large Globose Med Red 8-1-99-5B Ling Ping Yau sdlg. XL Pyriform Med Red HBJL-11 Hirado Buntan pink XL Globose Med Red 5-4-99-7-S2 Florida pummelo Large Globose Med/thin White 7-3-99-2 Siamese sweet Large Obovoid Med White 5-1-99-2-S5 Hirado Buntan pink XL Globose Thin Red 5-4-99-1-S7 Florida pummelo XL Obovoid Med White 8-1-99-1A Ling Ping Yau sdlg. Large Obovoid Med White

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 OLL-8 For more information on OLL-8 or OLL-4, please contact Florida Foundation Seed Producers, Inc. www.ffsp.net OLL-4 Variety Juice Quality Data from OLL-Series Somaclones Lbs. Juice Per Box Data 3-2-2012 Acid Total Brix Ratio Lbs. Solids Per Box Color Valencia Control 54.41 1.00 12.03 12.03 6.55 38.60 OLL 20 53.23 0.84 12.10 14.40 6.44 40.50 OLL 27 54.99 0.84 12.34 14.69 6.79 39.80 OLL 25 57.58 0.79 12.81 16.22 7.38 40.20 OLL 9 56.30 0.94 12.97 13.80 7.30 39.90 OLL 15 49.48 0.99 14.56 14.71 7.20 39.50 OLL 2 51.55 0.77 12.77 16.58 6.58 41.00 OLL 3 54.52 0.98 12.92 13.18 7.04 40.30 OLL 4 55.52 0.92 12.60 13.70 7.00 40.70 OLL 7 57.51 0.94 13.20 14.04 7.59 41.20 Valencia control/sw 48.46 0.79 12.91 16.34 6.26 39.10 OLL 21 58.20 0.77 12.38 16.08 7.20 40.30 OLL 10 55.55 0.82 12.42 15.15 6.90 40.60 OLL 5 53.92 0.93 13.13 14.12 7.08 40.10 OLL 23 57.21 0.81 12.12 14.96 6.93 39.90 OLL 19 54.02 0.84 12.32 14.67 6.65 40.90 OLL 1 55.10 0.90 12.90 14.33 7.11 41.50 OLL 6 58.19 0.96 12.85 13.39 7.48 40.80 OLL 3-10 dwarf 53.80 0.80 12.51 15.64 6.73 41.20 OLL 8 56.43 0.77 11.99 15.57 6.77 40.70 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 2 nd 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 2014 6.5 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.

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.

Valquarius on Orange #15 tetrazyg rootstock just < 5 years at St. Helena, Dundee FL released as UFR-3 Candidate for ACPS

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 Scion Rootstock Lbs Solids/Box 2012 2013 2011 (35 mo.) Yield Boxes/Tree 2012 (47 mo.) 2013 (59 mo.) Cumulative Yield (Boxes) Tress With Symptoms as of March 2013 Number of Trees in Trial Percentage with HLB as of March, 2013 (5 years) 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 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% VERNIA R. LEMON* 3.67 na NS 0.78 na 0.78 12 18 67% 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 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 Number of Trees in Trial Trees Removed 2014 Percent Trees Removed due to Poor Performance UFR-16 46X31-02-13* 8 0 0% 46X31-02-5* 13 0 0% 68-1G-26-F4-P2 12 0 0% UFR-13 FG 1731 5 0 0% UFR-14 FG 1733 5 0 0% Orange 1804 18 0 0% Volk 20 0 0% Yellow 1800 11 0 0% 68-1G-26-F6-P20 17 1 6% Rough Lemon 18 1 6% 46X31-02-11* 15 1 7% 68-1G-26-F2-P12 10 1 10% Swingle 20 2 10% 46X31-02-S3* 19 2 11% Aqua 1803 19 2 11% White 1805 19 2 11% 68-1G-26-F4-P6 13 2 15% HBJL-2B (n)* 23 4 17% 48-OP-01 28 5 18% Cleo 16 3 19% Kuharske Carrizo 63 12 19% 46X31-02-S9* 15 3 20% Pink 1802 18 4 22% MG11 40 12 30% White 1801 11 4 36% 46X31-02-9* 18 3 38% Chandler A1-11* 8 3 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 Number of Trees Trees removed 2014 Percent Trees Removed due to Poor Performance Tetraploid 6058X6056-00-2* 19 0 0% A-MAC* 19 0 0% AMB+BENT* 20 0 0% SO+RPXSH 99-4* 26 0 0% SR+SH-99-11* 7 0 0% SR+SH-99-6* 6 0 0% UFR-17 Green 2* 16 0 0% UFR-3 Orange 15 43 0 0% WGFT+50-7 86 0 0% Blue 4 37 5 1% UFR-5 White 4 72 1 1% Purple 4 64 1 2% SO+CZO 265 4 2% Blue 9 30 1 3% Chang+Bent 34 1 3% UFR-1 Orange 3 60 2 3% Blue 1 69 3 4% Blue 2 24 1 4% Orange 13 50 2 4% Orange 18 45 2 4% UFR-2 Orange 4 70 3 4% White 1 24 1 4% UFR-4 Orange 19 128 6 5% AMB+5-1-99-2* 18 1 6% Orange 14 62 4 6% 6058X2071-01-02* 27 2 7% Orange 16 27 2 7% SO+50-7 45 3 7% Orange 1 24 2 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 Jensen 1, Noemi Halbrendt 1,2, Gennaro Fazio 3, Izabela Makalowska 4, Naomi Altman 5, Craig Praul 6, Siela N Maximova 7, Henry K Ngugi 1,2, Robert M Crassweller 7, James W Travis 1,2 and Timothy W McNellis 1* 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. Crosses of superior parents made at diploid and tetraploid levels 2. Seed harvested from crosses planted in bins of calcareous soil (ph=8), inoculated with P. nicotianae and P. palmivora (JH Graham) 3. Selection of robust seedlings based on growth rate, health and color (most don t make it!) 4. Transfer to 4x4 pots in commercial potting soil 5. Top of new tree goes for seed source tree production; remaining liner to the HLB screen 6. Hybrid liner is grafted with HLB-infected budstick of Valencia sweet orange; remaining rootstock top removed, forced flushing from HLBinfected sweet orange budstick 7. 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 2 nd 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 2 nd 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. Variety Lbs. Juice Per Box Acid Total Brix Ratio Fruit Ct Lbs. Lbs. Solids Solids Per BoxPer Box juice color Vernia 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 53.443 0.98 9.53 9.72 58 5.0931 5.09 35.4

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