Finding tolerance and/or resistance to huanglongbing in citrus relatives

Chandrika Ramadugu1, Ed Stover2, Manjunath Keremane3, Greg McCollum2, David Hall2, Mikeal Roose1, and Richard Lee3 1University of California, Riverside, CA 2USDA ARS, Ft. Pierce, FL 3USDA ARS, Riverside, CA Why should we be concerned about finding resistance/tolerance to HLB?

After Hurrican Irma, even lower: 50M Source: Wall Street Journal Background of the search for HLB resistance • In 2007, we began research to evaluate several Citrus genotypes for tolerance to HLB – Seed from “Core Collection” was collected in CA, shipped to FL • “Core Collection” = 40 citrus hybrids identified using SSR markers which represent about 85% of the genetic diversity which was identified using 23 SSR markers, Barkley et al, 2006 – Other seed which had been collected and which was available at Repository – Type genotypes, P. trifoliata, pummelo, etc – Planted in the field in spring 2009 – 65 Citrus accessions, 33 accessions belonging to 20 other closely related genera Background of the search for HLB resistance  Located at USDA ARS Picos Farm, severe HLB, canker, no vector control.  Plants were fertilized, watered but and received minimal pest management care.  Took observations, collected samples in March and October each year, testing samples by qPCR for multiple regions of HLB genome.  DNA from mature leaves, selected symptomatic leaves if they were present.

Site for field trial Planted in the field at USDA ARS, Fort Pierce, FL Observations on HLB, canker and leaf miner, plant growth parameters.

October 2009

October 2013 October 2013 October 2009  Categorized the plants into 8 groups.  We know that there is no clear cut resistance in Citrus. Did not want to make it into three categories-susceptible, moderate and resistant.  We wanted to capture the plants that are in between these clear cut categories. S R

Category 8 6Category 1

7 7 1 1 3 5 4 4 Susceptible 2 Resistant Glycosmis Category One Immune; Glycosmis pentaphylla (orange berry) (psyllid colonized on these), and Clausena excavata (pink wampee). Never able todetect Las over the 6 year period. Hesperethusa Category Two Resistant; No symptoms, LAS negative. Afraegle, Bergera, Casimiroa edulis (white sapota) (psyllid did not colonize), Eremocitrus, Naringi, Simmon’s trifoliate, Zanthoxylum (psyllid did not colonize on this), Murraya exotica. Low levels of Las occasionally detected, but did not establish. Eremocitrus is sexually compatible with citrus; Naringi crenulata (Hesperethusa) and Eremocitrus are graft-compatible with citrus.

COX2010 COX2011 COX2012 LAS2010 LAS2011 LAS2012 LAS_LJABI 23.16 23.71 18.60 40.00 40.00 40.00 40.00 21.09 18.88 22.09 40.00 40.00 40.00 40.00 19.57 21.76 18.59 38.17 34.86 40.00 40.00 16.81 40.00 18.36 38.40 40.00 40.00 40.00 17.79 24.49 19.65 38.17 35.92 40.00 40.00 Category Three Highly tolerant; Within an accession, some plants are susceptible, some plants have no LAS or symptoms. Includes accessions: Balsamocitrus daweii, Mesero lemon, Microcitrus australis (Aust. Round lime), Sydney hybrid (M. australis X M. australasica), Citrus halimii, Faustrimedin (XMicrocitronella), Microcitrus australasica (Aust. finger lime).

Sydney hybrid Australian finger lime (M. australis X M. australasica) Microcitrus australasica Category four Tolerant; recover from infection. Aegle, Aeglopsis, Hassaku (pummelo hybrid) , Balsamocitrus daweii, Citrus latipes (‘Khasi’ papeda), Microcitrus australis (Aust. round lime), Hawaiian Mock Orange (Murraya sp.), Orange jasmine (Murraya sp.). Microcitrus COX2010 COX2011 COX2012 LAS2010 LAS2011 LAS2012 LAS_LJABI 21.76 20.28 15.514 27.1 32.99 28.279 34.123 18.77 21.94 18.582 40 40 30.495 38.892 17.44 27.49 20.205 31.97 30.2 33.628 38.270 23.73 28.9 16.277 40 31.25 40.000 40.000 27.77 20.65 17.934 40 40 40.000 40.000 17.59 23.72 19.465 40 40 40.000 39.413 Murraya paniculata (Hawaiian Mock Orange)

COX2010 COX2011 COX2012 LAS2010 LAS2011 LAS2012 LAS_LJABI 17.04 30.75 19.60 40.00 40.00 35.34 40.00 21.42 28.31 16.08 38.89 40.00 40.00 40.00 25.76 37.77 22.00 40.00 40.00 40.00 40.00 25.78 19.33 19.14 40.00 34.65 40.00 40.00 Murraya 40.00 22.90 18.38 40.00 37.04 40.00 40.00 17.02 40.00 18.32 40.00 40.00 40.00 40.00 There is infection. Plant can ward it off. 16.86 23.65 19.21 28.04 40.00 40.00 40.00 16.40 40.00 20.91 28.51 40.00 40.00 40.00 Category Five Tolerant, delayed infection; characterized by delayed infection with Las, Only two accession: X639 (Citroncirus), and little leaf trifoliate (Poncirus).

COX2010 COX2011 COX2012 LAS2010 LAS2011 LAS2012 LAS_LJABI X Citroncirus 21.16 19.18 18.74 40.00 40.00 23.71 22.36 21.16 19.18 22.95 40.00 40.00 40.00 22.36 X639 17.74 21.89 18.37 33.26 40.00 25.37 27.72 25.52 21.46 18.41 36.91 35.69 25.72 27.87 (Delayed 27.27 21.56 18.93 32.13 35.96 25.94 31.55 24.40 16.28 40.00 25.99 28.17 symptoms) 18.40 22.20 17.35 40.00 29.79 35.85 40.00 21.16 19.18 22.95 40.00 40.00 40.00 40.00 21.16 19.18 18.74 40.00 40.00 23.71 40.00 22.81 21.73 17.85 40.00 40.00 40.00 40.00 Poncirus trifoliata COX2010 COX2011 COX2012 LAS2010 LAS2011 LAS2012 LAS_LJABI 22.85 21.76 15.91 40.00 40.00 26.34 31.85 Little leaf 22.31 19.37 14.93 31.62 33.85 28.37 29.41 16.75 24.67 18.71 40.00 30.29 29.32 32.62 (Delayed 16.32 40.00 16.70 40.00 40.00 29.66 31.47 symptoms) 24.40 40.00 18.58 40.00 29.13 30.06 28.70 22.13 40.00 15.10 40.00 38.19 30.60 35.12 33.54 18.63 16.99 40.00 40.00 40.00 40.00 14.58 28.74 18.63 37.26 40.00 40.00 40.00 Category descriptions

Category Symptoms Typical accession in category 6 Susceptible to 38 accessions, mostly belonging to Las Citrus, Severinia, and Xcitroncirus, consistently tested positive for Las, remained alive for 4 years but retained some leaves 7 Susceptible to 25 accessions including many Las mandarins, certain pummelos, sour oranges, some lemons and limes. Alive for 4 years but severe symptoms and leaf loss 8 Susceptible to Died before 4 years, included Las some mandarins Field trial in Florida identified HLB resistant and tolerant accessions. Plants that showed immune, resistant or tolerant response to citrus HLB in our field trial are listed here: Immune (no HLB multiplication): Clausena excavata and Glycosmis pentaphylla. Resistant (transient replication but HLB does not establish in the host): Bergera koenigii, Casimiroa edulis, Eremocitrus glauca, Murraya paniculata (Orange jasmine), Naringi crenulata, Zanthoxylum ailanthoides. Tolerant (lower level of HLB but plants survive and produce fruit): Citrus halimii, Microcitrus australasica, Microcitrus hybrid (‘Sydney hybrid’), M. inodora, ‘Faustrimedin’, ‘Simmon’s trifoliate’, Aegle, Aeglopsis, Balsamocitrus daweii, Citrus latipes, Microcitrus Publications: australis, ‘Hawaiian mock orange’, ‘Little leaf’ http://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-03-16-0271-RE trifoliate, ‘X639’ trifoliate. http://citrusresearch.org/wp-content/uploads/CRB-Citrograph-Mag-Q2-2016-web.pdf (pp 46-51) Accessions that are sexually compatible with Citrus are indicated in red font with underline. These may be useful in breeding trials. HLB resistant and tolerant Australian limes selected for breeding Breeding for resistance: List of successful crosses

No. Seed parent Pollen parent 1 Australian Finger lime C146 trifoliate 2 Australian Finger lime Carrizo 3 Australian Finger lime Eremolemon 4 Australian Finger lime Hirado Buntan pummelo 5 Australian Finger lime Simmons trifoliate 6 Encore mandarin Australian Desert lime No. Seed parent Pollen parent 7 Encore mandarin Australian Finger lime 19 Fortune mandarin Australian Round lime 8 Encore mandarin Eremolemon 20 Fortune mandarin Large leaf Australian wild lime 21 Fortune mandarin Sydney hybrid 9 Encore mandarin Large leaf Australian wild lime 22 Monoembryonic trifoliate Australian Desert lime 10 Encore mandarin Sydney hybrid 23 Pomeroy trifoliate Australian Desert lime 11 Fallglo mandarin Australian Desert lime 24 Pomeroy trifoliate Australian Round lime 12 Fallglo mandarin Australian Finger lime 25 Pomeroy trifoliate Large leaf Australian wild lime 13 Fallglo mandarin Eremolemon 26 Rich 16-6 trifoliate Australian Finger lime 14 Fallglo mandarin Large leaf Australian wild lime 27 Temple tangor Australian Desert lime 15 Flying Dragon trifoliate Australian Finger lime 28 Temple tangor Australian Finger lime 16 Flying Dragon trifoliate Australian Round lime 29 Temple tangor Eremolemon 17 Flying Dragon trifoliate Large leaf Australian wild lime 30 Webber-Fawcett trifoliate Australian Finger lime 18 Fortune mandarin Australian Finger lime 31 Volckameriana lemon hybrid Australian Desert lime 32 Wilking mandarin Australian Desert lime 33 Wilking mandarin Australian Finger lime 34 Wilking mandarin Australian Round lime 35 Wilking mandarin Eremolemon 36 Wilking mandarin Large leaf Australian wild lime 37 Wilking mandarin Sydney hybrid Resistance observed in citrus relatives is heritable We determined that: 1. There is resistance in the sub family Aurantioideae (field trial). 2. The resistance is heritable (controlled greenhouse experiments).

X

Eremocitrus glauca ‘Meyer lemon’ Plant no. 69 Plant no. 95 Ct for Las is 40. Ct for Las is 26. HLB resistant. HLB susceptible. ‘Eremolemon’ Plant no. 93 Ct for Las is 40. HLB resistant. Experiments in the greenhouse were under controlled conditions (no-choice psyllid feeding). Resistance observed in the hybrid. How to distinguish hybrids? 1. Morphological difference can be observed in most hybrids

Microcitrus australasica Wilking X Pollen parent Microcitrus australasica 2. When necessary, sequence a part of the genome and compare with the expected sequence. Mandarin parent (No Microcitrus) GAP GAP Microcitrus parent (No Mandarin)

Mandarin Microcitrus hybrid

GAP Steps involved: 1. Conduct crosses. 2. Confirm hybrid nature of seedlings. 3. Challenge with psyllids in Florida greenhouses and in the Contained research facility in Davis, CA. Psyllid challenge 4. (Future) Evaluation of HLB resistance and genetic characterization  Our approach to develop resistance is unique since we are trying to transfer resistance from distant citrus relatives in the subfamily Aurantioideae from Australia. The genera Microcitrus (Australian finger lime) and Eremocitrus (Australian desert lime) are separated from Citrus by about 3.5 million years (Schwartz et al., 2015) and may have developed different mechanisms of resistance.

 Development of resistant Citrus by either traditional or transgenic means appear to be the ideal long term solution not only for sustainable production, but for reduction of pesticide usage, environmental benefits include protection of beneficial insects like honey bees. HLB resistant hybrids may be useful as rootstocks, inarches (approach grafts) or as interstocks to impart resistance to currently used rootstocks and scions.

Genotyping of HLB resistant and susceptible hybrid populations may be useful in identifying markers associated with HLB resistance.

Citrus with HLB resistance would lead to reduced management costs, higher level of production and a sustainable citrus industry. Reduced pesticide usage would lead to immense environmental benefits by reducing pollution, and allowing beneficial insects. Thank you for your attention

Richard Lee [email protected]