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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Insects and Horticulture Research » Research » Research Project #425052

Research Project: Genetic Improvement of Citrus for Enhanced Resistance to Biotic and Abiotic Stresses

Location: Subtropical Insects and Horticulture Research

2018 Annual Report

1. Create new genetic combinations of citrus germplasm via conventional breeding, mutation, and transformation, to include rootstock and scion development and evaluation for essential traits of disease resistance and horticultural qualities. 1.A. Use sexual hybridization to create new germplasm from diverse parental types with useful horticultural characteristics. 1.B. Create new scions with useful traits through mutation. 1.C. Create new scions and rootstocks with potential resistance to huanglongbing and citrus bacterial canker by genetic transformation. 2. Develop and evaluate methods to improve citrus transformation, including the use of proliferating in vitro shoot cultures, as a novel source for genetic transformation and germplasm preservation. 2.A. Develop methods to produce proliferating in vitro shoot cultures of rootstock and scion types. 2.B. Determine the transformation efficiency of in vitro shoot cultures. 3. Develop and evaluate new methods to efficiently screen germplasm for important traits, improve the process of citrus variety development, and apply appropriate methods to select superior individuals. 3.A. Refine and evaluate methods to assess huanglongbing tolerance/resistance, and apply appropriate methods to select superior individuals. 3.B. Develop and apply methods to test selections for abiotic stress, including high pH. 4. Evaluate field performance and other traits for rootstock and scion selections and release new cultivars as appropriate.

New citrus selections will be created by sexual hybridization, mutation, and genetic transformation from existing cultivars and species. Sources of tolerance or resistance to huanglongbing will be emphasized in choice of parents for hybrids. Genes with potential to induce tolerance or resistance to huanglongbing will be emphasized in transformation, including anti-microbial peptides, chimeral anti-microbial peptides, citrus genes that respond to infection by the pathogen, but with regulation altered to increase resistance, and genes that target specific metabolic components of the pathogen. Methods will be developed to improve citrus transformation, including the use of proliferating in vitro shoot cultivars. Hybrids and other new types will be assessed for important traits, including the use of molecular markers, and greenhouse, laboratory, and field assays. Methods to assess huanglongbing tolerance or resistance and tolerance of high pH will be refined and applied to new hybrids and transgenics. Promising selections will be entered into long-term field trials at multiple locations, and data will be collected on tree health, size, fruit yield and quality. Selections that appear to have desirable combinations of traits will be released for commercial or dooryard use.

Progress Report
This is the fifth and final year of a project that continues the long-term goals of previous projects to develop new citrus scion and rootstock cultivars with traits critical for successful commercial production and marketability. This project was replaced by project 6034-21000-018-00D, "Genetic Improvement of Citrus for Enhanced Resistance to Huanglongbing Disease and Other Stresses." The current project involves new work, especially focused on developing new rootstocks and scions resistant or tolerant to the disease huanglongbing. Toward this goal, over the past five years the project has created and begun testing on thousands of new hybrids with potential outstanding traits, including resistance to huanglongbing. Under the project, five new rootstocks and four new scions were released with evidence for improved field tolerance or resistance to huanglongbing. In addition, under subordinate externally-funded projects, research was completed to document gene expression, metabolomic, physiological, and anatomical differences among genotypes that is associated with disease sensitivity or tolerance, and to explore new avenues to develop huanglongbing resistant cultivars. In 2017-18, forty new hybrids were selected from large populations for further evaluation as rootstocks. Data was collected on tree size, health, and cropping from ten established replicated rootstock and field trials. Four new replicated rootstock field trials were planted, to evaluate 150 new rootstock hybrids. Trees were prepared for planting of seven additional rootstock field trials in 2018-2019, including about 100 additional new hybrid rootstocks. Work expanded under material transfer agreements and externally-funded grants to cooperatively test new scions and rootstocks in field trials with university and private partners in Florida, and in other states. Information was assembled to support the 2018 release of three new hybrid rootstocks with field tolerance to huanglongbing and outstanding field performance in graft combination with commercial scions. Greenhouse studies were conducted to evaluate rootstock tolerance to biotic and abiotic stress, including focused studies of shoot and root responses to controlled infection with the bacteria causing huanglongbing, and measuring seasonal titer of the bacteria as affected by tolerant cultivars. Citrus stage 1 and stage 2 shoot cultures have been developed and are being used in various aspects of citrus research, particularly those relating to huanglongbing. Psyllids feed and do well on Stage 1 and 2 shoot cultures. This finding opens up a number of potential applications for the use of these cultures in research. Eliminating unwanted fungal and bacterial contamination from these cultures is extremely difficult. Disinfestation treatments are being developed for this system. Stage 2 shoot cultures of some commercially produced citrus rootstocks have been developed as established proliferating shoot cultures suitable for mass propagation. These include US-942, Carrizo, US-897, X-639, and US-1516. Additionally, huanglongbing-infected stage 2 cultures are being developed from citron, a model citrus system for research. Huanglongbing-positive citron cultures are partially adapted to culture but have not yet transitioned to stage 2 growth. Experimentation to shorten this period is ongoing, but this effect is very poorly documented in the literature. Advances continue in developing improved conventional citrus scions. Over 14,000 new scion program hybrids have been planted in the last eight years. As our understanding of huanglongbing (HLB)-tolerance advances we make “better” crosses each year. Outstanding fruit quality and early evidence of huanglongbing-tolerance is evident in many populations. Potentially useful huanglongbing-tolerance has been found in some existing cultivars. Huanglongbing-resistance has been found in other genera within the citrus gene pool. A large replicated trial of 50 advanced selections and cultivars is in the fifth year of severe huanglongbing challenge and a number of our selections are showing excellent growth despite huanglongbing pathogen-infection. The best performer is a full sib of our best mandarin selection for which data to support a patent is being collected. A marked decline in growth rate appears to be an early indicator of low huanglongbing-tolerance in the worst performers. Eight citrus scion cultivars have been released in the last six years and more are in the pipeline including the first scion for use as a fresh fruit containing Poncirus in the pedigree. Large replicated plantings continue to be phenotyped, in an effort to identify genes associated with huanglongbing-tolerance and huanglongbing-resistance. In a multi-institution collaboration, specific gene markers were identified associated with huanglongbing-tolerance in Poncirus hybrids. Expression of huanglongbing pathogen effectors in citrus leaves has been shown to be evident within a few hours of psyllid inoculation and the prominent effectors expressed may provide early indications of tolerance and/or resistance. Progress continues in developing transgenic citrus with huanglongbing-resistance. A patent was received for a modified plant thionin (Mthionin) which greatly reduces Candidatus Liberibacter asiaticus (CLas) when transgenic vs. wild-type rootstocks are grafted with infected scions (1800X reduction at 12 months). Initial replicated trees have been placed in field planting, many additional Mthionin Carrizo and Hamlin have been propagated, and Florida Department of Agriculture, has received buds for cleanup, which are the first transgenic citrus they have received from any program. Transgenics expressing chimeral peptides (containing separate lytic and gram negative membrane recognition sequences from citrus) have been created and show greatly suppressed huanglongbing pathogen in detached leaf psyllid inoculations. A Cooperative Research and Development Agreement has been initiated to develop the data package to deregulate Mthionin transgenics. Small trees transgenically expressing antibodies to two exterior huanglongbing-pathogen proteins were exposed to huanglongbing pathogen in no-choice psyllid feeding. In these studies, pathogen suppression was 400x after nine months. A phloem-specific promoter from citrus is being used in creating transgenics targeting the huanglongbing pathogen. New transgenics have also been created expressing peptides or double-stranded ribonucleic acids identified by collaborators to prevent huanglongbing pathogen acquisition by Asian citrus psyllid. Advances have been made in developing methods to expedite screening for genotype response to huanglongbing pathogen and rapid screening of potential transgenic products and other therapies. We have shown that detached leaves from transgenics can be used in psyllid inoculation assays to verify Liberibacter killing activity. Disks punched from infected leaf petioles and infected Asian citrus psyllid homogenates are showing promise as methods to screen peptides for pathogen-clearance, that may be expressed transgenically, as well as potential pathogen-eliminating therapies.

1. Trangenic citrus suppresses huanglongbing pathogen. Within citrus germplasm there is a range of tolerance and some partial resistance to the pathogen causing huanglongbing disease. However, there seems to be no strong resistance to this pathogen within the citrus gene pool. ARS researchers at Ft. Pierce, Florida, have demonstrated efficacy of some transgenics in suppressing the huanglongbing pathogen. Transgenics expressing a modified thionin or chimeral peptides (containing separate lytic and gram negative membrane recognition sequences from citrus) were shown to greatly suppress huanglongbing-pathogen in detached leaf psyllid inoculations and/or potted plant inoculations. Small trees transgenically expressing antibodies to two exterior pathogen epitopes were exposed to no-choice feeding by psyllids, and huanglongbing-pathogen suppression was 400x compared to controls after nine months. These transgenics are propagated for field testing of huanglongbing resistance, and provide hope that fully resistant cultivars will be available for future use.

2. Established huanglongbing-infected shoot cultures. Studying huanglongbing in the greenhouse and field is difficult and expensive because of citrus biology (long juvenile period, inbreeding depression, very inefficient scion genetic transformation methods, and as a tree crop requires substantial greenhouse and field infrastructure for experimentation), and long times required to test and validate potential control and treatment strategies. The pathogen causing huanglongbing, Candidatus Liberibacter asiaticus, cannot be cultured. The use of in vitro shoot culture for huanglongbing research has the potential to provide new methods that are less costly and more rapid than current greenhouse- and field-based approaches. ARS researchers at Ft. Pierce, Florida, have successfully cultured infected shoots in vitro through multiple cycles of culture. After 8 months the shoots remain infected. The shoots exhibit disease symptoms (leaf mottling), but are sufficiently healthy that it appears stage 2 proliferating cultures might be possible. Stage 2 cultures are in vitro plants that grow and multiply sufficiently for mass propagation. Such HLB-infected in vitro cultures have never been developed but could be quite useful for a variety of applications.

3. Efficient citrus rootstock propagation by cuttings. Conventional citrus rootstock propagation is by seed, but sufficient seed is not available for many of the best disease-resistant rootstocks needed by the citrus industry. No seed is available for some of the newest rootstocks in high demand. ARS researchers at Ft. Pierce, Florida, developed methods that are readily applied in commercial nurseries, to efficiently propagate citrus rootstocks by vegetative cuttings. These methods are being applied on large scale in Florida commercial nurseries. The use of cuttings for effective citrus rootstock propagation allows much more rapid testing and release of new rootstocks by researchers, and allows large scale commercial propagation of rootstocks where adequate seed is not available. This markedly accelerates adoption of new and better rootstocks into commercial use.

4. Minnie Finger lime. Numerous niche markets exist for specialty citrus fruit, and one such market is for finger lime, a citrus-type fruit that tastes like a lime and is shaped like a finger. Fruit of the finger lime is highly valued in restaurants for use as a garnish and flavoring for food, sometimes called “citrus caviar”. Finger lime cultivars have previously not been identified that produce well in Florida. ARS researchers at Ft. Pierce, Florida, developed and released a new cultivar, Minnie Finger lime. This cultivar has the characteristics of finger lime, and appears productive under Florida conditions, and presents the opportunity for commercial production of finger lime in Florida.

Review Publications
Hao, G., Zhang, S., Stover, E.W. 2017. Transgenic expression of antimicrobial peptide D2A21 confers resistance to diseases incited by Pseudomonas syringae pv. tabaci and Xanthomonas citri, but not Candidatus Liberibacter asiaticus. PLoS One. 12(10):e0186810.
Dasgupta, K., Thilmony, R.L., Stover, E.W., Oliveira, M.L., Thomson, J.G. 2017. Novel R2R3-MYB transcription factors from Prunus americana regulate differential patterns of anthocyanin accumulation in tobacco and citrus. GM Crops & Food. 8:85-105.
Pisani, C.N., Ritenour, M.A., Stover, E.W., Plotto, A., Alessandro, R.T., Kuhn, D.N., Schnell II, R.J. 2017. Postharvest and sensory evaluation of selected ‘Hass’x‘Bacon’ and ‘Bacon’x ‘Hass’ avocado hybrids grown in East-Central Florida. HortScience. 52(6):880-886. https://doi:10.21273/HortSci.11375-16.
Hall, D.G., Hentz, M.G., Stover, E.W. 2017. Field survey of Asian citrus psyllid (Hemiptera: Liviidae) infestations associated with six cultivars of Poncirus trifoliata. Florida Entomologist. 100:667-668.
Belknap, W.R., Thomson, J.G., Thilmony, R.L., McCue, K.F., Hao, G., Stover, E.W. 2017. Small cyclic amphipathic peptides (SCAmpPs) genes in citrus provide promising tools for more effective tissue specific transgenic expression. Acta Horticulturae. 1172:85-90.
Volk, G.M., Samarina, L.K., Kulyan, R., Vyacheslav, G., Malyarovskaya, V., Ryndin, A., Polek, M., Krueger, R., Stover, E.W. 2017. Citrus genebank collections: International collaboration opportunities between the U.S. and Russia. Genetic Resources and Crop Evolution. 65:433-447.
Zhang, S., Shi, Q., Albrecht, U., Shatters, R.G., Stange Jr, R.R., McCollum, T.G., Zhang, S., Fan, C., Stover, E.W. 2017. Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants. Horticulture Research. 4:17041.
Collier, R.A., Dasgupta, K., Xing, Y., Hernandez, B., Shao, M., Rohozinski, D., Kovak, E., Lin, J.W., De Oliveira, M., Stover, E.W., Mc Cue, K.F., Harmon, F.G., Blechl, A.E., Thomson, J.G., Thilmony, R.L. 2017. Accurate measurement of transgene copy number in crop plants using droplet digital PCR. Plant Journal. 90:1014-1025.
Collier, R.A., Xing, Y., Lin, J.W., Mc Cue, K.F., Blechl, A.E., Thomson, J.G., Thilmony, R.L., Dasgupta, K., Hernandez, B.T., Shao, M., Oliveira, M.L., Stover, E.W., Novak, E., Harmon, F.G., Rohozinski, D. 2017. Accurate measure of transgene copy number in crop plants using droplet digital PCR. Plant Journal. 9(5):1014-1025 doi: 10.1111/TPJ.13517.
Bowman, K.D., Albrecht, U. 2017. Efficient propagation of citrus rootstocks by stem cuttings. Scientia Horticulturae. 225:681:688.
McCollum, T.G., Bowman, K.D. 2017. Rootstock effects on fruit quality among 'Ray Ruby' grapefruit trees grown in the Indian River district of Florida. HortScience. 52(4):541-546. https://doi:10.21273/HortSci.11435-16.
Akin, M., Eyduran, E., Niedz, R.P., Reed, B.M. 2017. Developing hazelnut tissue culture medium free of ion confounding. Plant Cell Tissue and Organ Culture. https://doi:10.1007/s11240.017-1238z.
Huynh, M.P., Meihls, L.N., Hibbard, B.E., Lapointe, S.L., Niedz, R.P., Ludwick, D.C., Coudron, T.A. 2017. Diet improvement for western corn rootworm (Coleoptera: Chrysomelidae) larvae. PLoS One. 12(11):e0187997.
Lapointe, S.L., Barros-Parada, W., Fuentes-Contreras, E., Herrera, H., Kinsho, T., Miyake, Y., Niedz, R.P., Bergmann, J. 2017. Use of mixture designs to investigate contribution of minor sex pheromone components to trap catch of the carpenterworm moth, Chilecomadia valdiviana. Journal of Chemical Ecology. 43(11-12):1046-1055.