HLB Resistance and Tolerance in Citrus Scion Breeding

HLB Resistance and Tolerance in Citrus Scion Breeding

Report Date: 04/14/2017
Project: 15-025   Year: 2017
Category: Horticultural & Management
Author: Ed Stover
Sponsor: Citrus Research and Development Foundation

1) Plants were generated after exposure of buds to several antibiotic levels to generate a range of infections and plants were monitored for HLB development, CLas, and growth. Within each genotype there was no correlation between final size after 42 months and the CLas titer. The documented tolerant ‘Jackson’ grapefruit and the grapefruit-like selection Ftp 6-17-48 displayed significantly greater stem diameter than all other genotypes. 2) Trees have been in the ground for over three years in a trial of 50 selections and cultivars following no-choice ACP inoculation and several months in an ACP house. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. The trees looking healthiest include a full sib of our best mandarin selection, and several of our best grapefruit-like hybrids. The one true grapefruit is the least healthy selection in the trial. It may take 2-3 more years to clearly distinguish tolerant material. 3) In June 2015 a field planting was established of: seedling trees of 133 Fortune x Fairchild hybrids from an earlier mapping study, seedlings of 27 Ponkan-like accessions, budded trees of 10 advanced ARS selections that are predominately mandarin, and budded trees of Fortune, Fairchild and Valencia. Data collection is underway. 4) Trials are established. Volatiles from sweet-orange-like hybrids were shown to be so similar to sweet orange that likely can be legally designated as sweet orange. 5) RNA-seq compared transcriptome responses in HLB moderately tolerant �Sun Chu Sha� mandarin and susceptible �Duncan� grapefruit, to Xcc-flg22 and CLas-flg22 (project initiated with Gloria Moore at University of Florida). Recently data analysis revealed that a group of 86 genes were differentially regulated by CLas-flg22 in �Sun Chu Sha� mandarin but not in �Duncan� grapefruit and not associated with differential expression from Xcc-flg22, suggesting they may have roles in HLB tolerance. The 16 genes with highest differential expression were selected for RT-qPCR validation, and 10 genes were consistent with the RNA-seq results. To evaluate if these genes were associated with HLB tolerance, �Cleopatra� mandarin (similar to �Sun Chu Sha�) and �Duncan� grapefruit plants were inoculated with CLas using psyllid infestation. CLas titer and gene expression were monitored biweekly for 10 weeks after inoculation. High bacterial titer (Ct<30) was observed at 2 weeks in �Duncan� but not until 6 weeks in �Cleopatra�. RT-qPCR results indicated that 5 of the studied genes were differentially expressed between the �Cleopatra� HLB-infected and the uninfected control plants, but not in �Duncan�. Induction of these genes was detected before bacterial infection was detected. Although not fully annotated in the citrus genomic databases, the function of some of these genes include a peroxidase, gibberellin 2-beta-dioxygenase, glucan endo-1,3-beta-D-glucosidase and an F-box domain containing protein. We will continue to characterize the expression of these genes and their association to HLB tolerance in other citrus genotypes, and determine if they may serve as marker genes for selection of tolerant citrus material. 6) Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, are being grown, and will be planted in the spring for field testing of HLB resistance. 7) Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance is completed, has revealed potentially valuable tolerance and indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. In August 2010, the plants were established at Pico�s farm in Ft. Pierce FL. Despite the high incidence of mottle in �SugarBelle�/ SourOrange, it had the greatest overall increase in diameter. 'SugarBelle' and 'Tango' (which were not on the same stock as 'Hamlin' and so results should be viewed as comparing cultivar/rootstock combinations) were the healthiest in overall appearance in 10/15 and had the most fruit (88 per tree). 8) Data are being collected which will contribute to the objective of assessing economic value of tolerance 9) This objective focuses on rapid HLB-resistance screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees that are exposed to no-choice CLas infected ACP feeding. CLas titer levels, using real time PCR, are easily detectable in most plants at 3 weeks. By nine weeks after exposure, susceptible genotypes can be clearly distinguished from reported resistant material by higher CLas levels in roots. Averaged across genotypes and tissues, total CLas per tree was 5 billion in week 3 after ACP exposure and doubled every 3 weeks through week 12. This should be especially useful for screening anti-Las transgenics. 10) Our putative chimeras have not proven to be successful. We identified a chimera (Satsuma and Poncirus) from the Citrus genebank, arranged its importation, and have diligently communicated with DPI to get it released to us. Shoot-tip-grafting has not been successful, but we finally got permission to accept this material and maintain it in a quarantine death house. 11) Initial cuttings have been made.


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