Progress 05/01/13 to 04/30/18
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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.
Impacts
(N/A)
Publications
- Hao, G., 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. Biomed Central (BMC) Plant Biology.
- 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.
- 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. 10.1007/2107-017-0543-z.
- 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.
- 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.
- 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. https//:doi.org/10.1007/s10886.017. 0906-0..
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. This is the fourth 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. The current project involves new work, especially focused on developing new rootstocks and scions resistant or tolerant to the disease huanglongbing. Data on tree size, health, and cropping was collected from ten established rootstock and scion field trials. Three new replicated field trials were planted. Trees were prepared for planting of ten additional rootstock field trials in 2017-2018. Work expanded under material transfer agreements to cooperatively test new scions and rootstocks in field trials with university and private partners. Cross hybridizations for improved scions and rootstocks were conducted and new hybrid seedlings were planted in the greenhouse. Material was selected from existing promising greenhouse and field trees for further study. Greenhouse and field studies continued to assess rootstock and scion tolerance of Candidatus Liberibacter asiaticus infection and huanglongbing disease. Greenhouse and field work indicate highly significant effects of rootstock and scion genotype on tree tolerance or resistance to huanglongbing, the disease caused by Candidatus Liberibacter asiaticus. Over 11,000 new scion program hybrids have been planted in the last seven years, with crosses from 2011 and 2012 starting to fruit. These are the first hybrids created specifically to target resistance/tolerance to huanglongbing. As our understanding of huanglongbing-tolerance advances we make better crosses each year. For example, we are now actively using breeding parents with citron in their pedigree, reflecting new data indicating citron confers tolerance. Outstanding fruit quality and early evidence of huanglongbing-tolerance is evident in many populations. A large replicated trial of 50 advanced selections and cultivars is in the fourth year of severe huanglongbing challenge and a number of our selections are showing excellent growth despite infection. Large replicated plantings of our most-advanced selections were planted at five grower sites, and trees are being prepared for very large scale replicated trials using funds from a grant. Large replicated plantings continue to be phenotyped, in an effort to ultimately identify genes associated with huanglongbing tolerance and resistance. Advances have been made in developing methods to expedite screening for genotype response to huanglongbing and rapid screening of potential transgenic products and other therapies. In FY 17, transient intermittent water- or nutrient-stress was shown to accelerate huanglongbing development in graft-inoculated trees by two months compared to non- stressed trees. Interestingly, the greatest stress treatment eliminated the pathogen over time. Infected detached leaves and disks punched from infected leaf petioles are showing promise as methods to screen peptides for elimination of the pathogen that may be expressed in transgenic plants, or as a therapy for eliminating the pathogen in existing trees. Progress continues in developing transgenic citrus with huanglongbing- resistance. In the previous year, transgenic Carrizo rootstock plants expressing a modified plant thionin (Mthionin) were grafted with scions infected with the huanglongbing-pathogen and after one year the control Carrizo roots had 1800 X higher pathogen titer than the transgenic Carrizo. A patent application has been completed on Mthionin. Hamlin, Valencia, and Ray Ruby transgenics expressing this peptide have been created. Replicated trees have been prepared for field planting using the Mthionin Carrizo and non-transgenic scions. Transgenics expressing chimeral peptides (containing separate lytic and gram negative membrane recognition sequences) have been created combining Mthionin or citrus thionin with a citrus lipid binding protein. Small trees have been exposed to no-choice feeding by Candidatus Liberibacter asiaticus- infected Asian citrus psyllid, and effects on pathogen suppression will be determined. A promoter from the most highly expressed gene in citrus phloem has been characterized and is being used in creating transgenic plants targeting Candidatus Liberibacter asiaticus. Using a reporter gene, this promoter (396ss) had 500x greater messenger Ribonucleic acid (RNA) in the midrib compared to the laminar area, vs. identical laminar and petiole expression from a more common promoter. The 396ss promoter is being actively used to create transgenic citrus and a patent application for 396ss is in development. New transgenic plants have also been created expressing peptides identified by ARS researchers in Ft. Pierce, Florida, as preventing Candidatus Liberibacter asiaticus acquisition in Asian citrus psyllid. Citrus stage 1 and stage 2 in vitro shoot cultures have been developed for some important citrus rootstock types � Carrizo, X-639, and US-942. Plant in vitro culture recognizes 5 stages as follows: 1) Stage 0 � Preparative stage. Mother plants are grown and treated to minimize microbial contamination and maximize the in vitro response of the explants. 2) Stage 1 � Initiation of axenic culture stage. Axenic cultures are established followed by some growth. 3) Stage 2 � Multiplication stage. The cultures produce sufficient shoots for subsequent propagation (or source material for transformation) as well as shoots to maintain the stock. Note: There are few reports that clearly describe this stage in citrus. 4) Stage 3 � Preparation for ex vitro growth stage. Shoots are prepared, generally elongated, for in/ex vitro rooting or grafting (commonly done in citrus). 5) Stage 4 � Transfer to ex vitro growth stage. Identify the conditions required to transition an in vitro plant to ex vitro growth (e.g, greenhouse). Identifying the appropriate conditions is important since significant losses can occur at this stage. Stages 1 and 2 have not been used in citrus breeding and genetics but would be quite useful in citrus for a number of applications including genetic transformation. Many of the advantages of using these sorts of cultures is that they are both clonal and vegetative; thus, genetic transformation could be applied to all citrus types, not just those that produce polyembryonic seed (the only citrus types now transformed). One observation from this research is that the physiological transition from a stage 1 culture to a stage 2 culture takes from 1 to 2 years. This is a very long time, and something not expected or reported in the literature. A Carrizo culture was developed that took 13 months to adapt. We have a citron culture that is now 13 months old and still has not adapted. Most plant species adapt in under 3 months. We now have three citrus rootstock varieties established as stage 2 cultures � Carrizo, X-639, and US-942. Citron is being developed as a model system because of its high susceptibility to huanglongbing (HLB), and thus it�s potential as a useful model in HLB research. Psyllid infestation was attempted a number of times but the resulting contamination could not be eliminated. Psyllids readily fed on in vitro shoots and laid eggs that resulted in larva. Further, electrofeeding experiments were conducted to determine how feeding on in vitro shoots compared to feeding on ex vitro feeding. A mineral nutrition software tool was developed. ARS-Media for Excel was developed to solve mineral nutrition linear programming equations. An Excel application solves the mineral nutrient linear programming calculations required for in vitro mineral nutrition research for improving the growth of in vitro shoot cultures. Use of this software is required for all mineral nutrient experiments that involve the culture of organisms (such as citrus and huanglongbing (HLB)). Accomplishments 01 Superior multi-year field performance for two USDA citrus rootstocks. Many existing rootstocks decline rapidly when affected by huanglongbing disease, and this has become a devastating problem for the Florida citrus industry. Results from long-term field trials conducted by ARS researchers and affected by huanglongbing, indicate that the rootstocks US-802 and US-942 provide improved yield, fruit quality, and tree health as compared with standard commercial rootstocks. Both rootstocks are commercially available in limited quantities and ARS researchers are working with nurseries and micropropagation companies to expand production of the rootstocks to meet the large commercial demand. The results suggest that use of these or other tolerant rootstocks will be a key component of successful citrus production management in the presence of huanglongbing disease. 02 Identified sweet-orange-like hybrids as breeding parents. These hybrids are very similar to sweet-orange in flesh quality, produce only hybrid seed, have high gamete viability, produce sweet-orange-like progeny, and have little inbreeding depression in crosses with parents having a high proportion of sweet-orange in their pedigrees. Conventional sweet orange is a very poor breeding parent due to high levels of apomixis (which results in seedlings that are genetically identical to parents, and not hybrids) and also have a very narrow genetic base, making Florida citrus essentially a mono-culture. Three of these new selections have been released for use as sweet-orange breeding parents. These should contribute to enhanced breeding of sweet- orange types around the world and will contribute to developing citrus with sweet-orange traits but greater disease resistance. 03 Established stage 1 and stage 2 in vitro cultures. Three citrus rootstocks Carrizo, X-639, and US-942 were established as stage 1 and stage 2 cultures. Citron and Valencia sweet orange were established as stage 1 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. Also, the pathogen that causes huanglongbing, Candidatus Liberibacter asiaticus, cannot be cultured. The use of in vitro shoot cultures for huanglongbing research will provide new methods that are less costly and more rapid than current greenhouse- and field-based approaches and will compliment these approaches.
Impacts
(N/A)
Publications
- Niedz, R.P. 2016. ARS-Media: A spreadsheet tool for calculating media recipes based on ion-specific constraints. PLoS One. 11(11):e0166025. doi:10.1371/journal.pone.0166025.
- Bowman, K.D., Faulkner, M.L., Kesinger, M. 2016. New citrus rootstocks released by USDA 2001-2010: field performance and nursery characteristics. HortScience. 51(10):1208-1214.
- Stover, E.W., Shatters, R.G., Gruber, B., Kumar, Moore, G.A. 2016. Influence of photoperiod duration and phloem disruption through scoring on growth, disease symptoms and bacterial titer in citrus graft-inoculated with Candidatus Liberibacter asiaticus. HortScience. 51:1215-1219.
- Stover, E.W., Lin, Y., Yang, X., Vashisth, T. 2016. Hydrogen cyanamide on citrus: preliminary data on phytotoxicity and influence on flush in potted and field trees. HortTechnology. 26:839-845.
- Miles, G., Stover, E., Keremane, M., Ramadugu, C., Lee, R.F. 2017. Apparent tolerance to huanglongbing in citrus and citrus-related germplasm. HortScience. 52:31-39.
- Keremane, M.L., Ramadugu, C., Halbert, S., Duan, Y., Roose, M., Stover, E. W., Lee, R. 2016. Long term field evaluation reveals HLB resistance in Citrus relatives. Plant Disease.
- Bai, J., Baldwin, E.A., Driggers, R.E., Hearn, J., Stover, E.W. 2016. Volatile and nonvolatile flavor chemical evaluation of USDA orange- mandarin hybrids for comparison to sweet orange and mandarin fruit. Journal of the American Society for Horticultural Science. 141(4):339-350.
- Pisani, C., Ploetz, R., Stover, E., Ritenour, M., Scully, B. 2015. Laurel wilt in avocado: Review of an emerging disease. International Journal of Plant Biology and Research. 3(3):1043-1049.
- Inch, S.A., Stover, E.W., Driggers, R.E., Lee, R.F. 2014. Freeze response of citrus and citrus-related genotypes in a Florida field planting. HortScience. 49:1010-1016.
- Hao, G., Pitino, M., Ding, F., Hong, L., Stover, E., Duan, Y. 2014. Induction of innate immune responses by flagellin from the intracellular bacterium, �Candidatus Liberibacter solanacearum�. Molecular Plant Pathology. 14:211.
- Niedz, R.P., Hyndman, S.E., Evens, T.J., Weathersbee, A.A. 2014. Mineral nutrition and in vitro growth of Gerbera hybrida (Asteraceae). In Vitro Cellular and Developmental Biology Plants. 50:458-470.
- Niedz, R.P., Albano, J.P., Marutani-Hert, M. 2015. Effect of various factors on shoot regeneration from citrus epicotyl explants. Journal of Applied Horticulture. 17(2)121-128.
- Hao, G., Stover, E.W., Gupta, G. 2016. Overexpression of a modified plant thionin enhances disease resistance to citrus canker and Huanglongbing (HLB). Frontiers in Plant Science. doi:10.3389/fpls.2016.01078.
- Rezzazadeh, R., Niedz, R.P. 2015. Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture- amount design. Plant Cell, Tissue And Organ Culture. 122(3):585-604
- Wada, S., Maki, S., Niedz, R.P., Reed, B.M. 2015. Screening genetically diverse pear species for in vitro CaCl2, MgSO4 and KH2PO4 requirements. Acta Physiologiae Plantarum. 37, 63 DOI 10.1007/s11738-014-1754-y.
- Wada, S., Niedz, R.P., Reed, B.M. 2015. Determining nitrate and ammonium requirements for optimal in vitro response of diverse pear species. In Vitro Cellular and Developmental Biology - Plants. 51(1):19-27.
- Niedz, R.P., Evens T. 2016. Design of experiments (DOE) - history, concepts, and relevance to in vitro culture. In Vitro Cellular and Developmental Biology Plants. 52:547-562.
- Mahmoud, S., Ramos, J.E., Shatters, R.G., Hall, D.G., Lapointe, S.L., Niedz, R.P., Rouge, P., Borovsky, D. 2016. Expression of Bacillus thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae. Pesticide Biochemistry and Physiology. 136:1- 11.
- Stover, E.W., Hall, D.G., Shatters, R.G., Moore, G.A. 2016. Influence of citrus source and test genotypes on inoculations with Candidatus Liberibacter asiaticus. HortScience. 51:805-809.
- Hall, D.G., Ammar, D., Bowman, K.D., Stover, E.W. 2017. Epifluorescence and stereomicroscopy of trichomes associated with resistant and susceptible host plant genotypes of the Asian citrus psyllid (Hemiptera: Liviidae). Journal of Microscopy and Ultrastructure. Available:
- Hummer, K.E., Dempewolf, H., Bramel, P., Markham, R., Stover, E.W. 2015. Status of global strategies for horticultural fruit crops. Acta Horticulturae. 1101:147-152. doi: 10.17660/ActaHortic.2015.1101.22.
- Belknap, W.R., Mc Cue, K.F., Harden, L.A., Vensel, W.H., Bausher, M.G., Stover, E.W. 2015. A family of small cyclic amphipathic peptides (SCAmpPs) genes in citrus. Genome. 16:303-313.
- Pascacio, C., Lapointe, S.L., Williams, T., Sivinski, J.M., Niedz, R.P., Aluja, M. 2014. Mixture-amount design and response surface modeling to assess the effects of flavonoids and phenolic acids on developmental performance of Anastrepha ludens. Journal of Chemical Ecology. 40:297-306.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. This is the third 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. The current project also initiates new work to address current threats and problems in United States citrus production, especially the disease problem huanglongbing. Cross hybridizations for improved scions and rootstocks were conducted and new hybrid seedlings were planted in the greenhouse. Material was selected from existing promising greenhouse and field trees for further study. Greenhouse and field studies continued to assess rootstock and scion tolerance of Candidatus Liberibacter asiaticus infection and huanglongbing disease. Greenhouse and field work, including several newly published studies, indicate highly significant effects of rootstock genotype on tree tolerance or resistance to huanglongbing, the disease caused by Candidatus Liberibacter asiaticus. The USDA rootstocks US-802, US-942, and US-1516 have appeared as the best rootstocks in field trials strongly affected by huanglongbing. Work also continued to assess rootstock tolerance to Citrus tristeza virus and calcareous soils. Data on tree size, health, and cropping was collected from twelve established rootstock and scion field trials. Five new replicated rootstock trials were planted in the field. Trees were prepared for planting of seventeen additional rootstock field trials in 2017. Work expanded under material transfer agreements to cooperatively test new scions and rootstocks in field trials with university and private partners. Grant-funded studies continue to produce and test transgenic scions and rootstocks, targeting increased tolerance to huanglongbing and canker diseases. Testing of selections for huanglongbing tolerance was conducted by controlled inoculation with the pathogen in the greenhouse using the vector Asian citrus psyllid, as well as field exposure under controlled conditions. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with the pathogen. Focused study continued on specific transgenics which show the most promise. Transgenic Carrizo rootstock expressing a modified plant thionin were grafted with scions infected with the huanglongbing-pathogen and after one year the control Carrizo roots had 1800 X higher pathogen titer than the transgenic Carrizo. Scion leaves on these small plants also showed markedly lower pathogen levels in transgenics compared to controls. Thionin-overexpressing transgenic scion cultivars have also been produced and are entering testing. Grant-funded studies continued to compare metabolic differences between citrus selections that differ greatly in their tolerance to huanglongbing. Tolerance to the disease did not appear to be associated with accumulation of higher amounts of protective metabolites in response to infection. Rather, the differences in tolerance appeared more likely associated with much higher constitutive expression and concentrations of protective metabolites in the tolerant cultivars. Provided field experimental design and statistical analysis for a project assessing potential mitigation of huanglongbing through application of antibacterial compounds. Data collected showed statistically significant improvement in tree health and pathogen titer reduction in most field plots, and some showed improved cropping. The need for improved citrus cropping is so great that these data provided support for a �Crisis Declaration of Use� by the Florida Ag Commissioner and the associated antibacterials have been applied to a majority of Florida citrus acreage. In collaboration with National Center for Genome Resources-Riverside (NCGR) a field planting of replicated seedlings from 85 diverse seed source genotypes, after seven years in the field, showed strong huanglongbing resistance in several genera related to citrus. In the same planting, substantial huanglongbing tolerance in the genus Citrus, as assessed by sustained growth and canopy health, was observed to be strongly associated with percentage citron in plant pedigrees. Experiments provided further documentation of tolerance to huanglongbing in conventional citrus scions exposed to high disease pressure from initial planting, with sustained growth and progression in cropping for three cultivars in a planting where sweet orange and grapefruit are in severe decline. 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. For example, because psyllids readily feed on in vitro plants, it may be possible to infect in vitro plants with huanglongbing to obtain a stable in vitro shoot culture containing the pathogen. Eliminating unwanted fungal and bacterial contamination from these cultures is difficult, and experimentation is ongoing to develop disinfestation methods. Currently, cultures with 24- hour psyllid infestation can be cleaned, but 7-day infestations cannot. Stage 2 shoot cultures are established proliferating shoot cultures suitable for mass propagation. Our intent is to use these cultures, and huanglongbing-infected stage 2 cultures, as source tissue for genetic transformation. Current stage 2 cultures are not quite as robust as they need to be for manipulation experiments. Currently, experimentation is ongoing to enhance the growth and vigor of these cultures. Also, stage 2 citrus cultures take well over a year to develop, and the process is poorly documented in the literature. Experimentation to shorten this period is ongoing. Accomplishments 01 Released one new citrus rootstock with tolerance to huanglongbing disease. ARS researchers at Ft. Pierce, Florida, identified and released one new citrus rootstock that exhibited much higher sweet orange fruit productivity and tree health in multiyear field trials growing in the Florida central ridge area severely affected by huanglongbing, as compared with commercial standard rootstocks. The new rootstock, US-1516 is available from the Florida clean budwood program, and is being propagated for entry into large scale grower trials in 2016-17. The results from field testing indicate that this rootstock, and US-942 rootstock that was previously released, enable sweet orange trees to better tolerate huanglongbing disease. Trees on these rootstocks become infected with the disease, but after infection these trees yield more fruit that have higher sugar content in comparison to orange trees on other standard rootstocks. The results suggest that use of these or other tolerant rootstocks will be a key component of successful citrus production management in the presence of huanglongbing disease. 02 Created transgenic citrus that suppresses huanglongbing pathogen. ARS researchers in Ft. Pierce, Florida, produced transgenic citrus strongly suppressing the huanglongbing pathogen. The disease huanglongbing is devastating the Florida citrus industry. Transgenic Carrizo expressing a modified plant thionin were grafted with scions infected with the huanglongbing pathogen and after one year the control Carrizo roots had 1,800 X higher pathogen titer than the transgenic Carrizo. Scion leaves on these small greenhouse plants also showed markedly lower huanglongbing pathogen levels in transgenics compared to controls. It may markedly enhance citrus production, if Florida growers can grow trees that suppress the huanglongbing pathogen. 03 Psyllids feed on in vitro citrus shoots. 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 cultered. 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 demonstrated that psyllids readily feed on stage 1 and stage 2 citrus and, that the feeding is primarily from the xylem. This is significant because psyllids are considered as phloem feeders in nature. One application would be to use the in vitro cultures to easily deliver molecules to the feeding psyllid (e.g., double-stranded Ribonucleic acid (dsRNA)). This may potentially make screening various molecules far more efficient then current methods. 04 Metabolic variations in different citrus rootstocks are associated with different responses to huanglongbing. Agriculture Research Service (ARS) researchers in Ft. Pierce, Florida, demonstrated that citrus selections which differ greatly in their tolerance to huanglongbing also have great large differences in the types and quantities of numerous biochemicals in shoot and root tissue. Some of these biochemicals or metabolites are known to play important roles in helping to protect host plants from other biotic or abiotic stresses. Tolerance to the disease did not appear to be associated with accumulation of higher amounts of protective metabolites in response to infection. Rather, the differences in tolerance appeared more likely associated with much higher constitutive expression and concentrations of protective metabolites in the tolerant cultivars. These observations provide critical insight to the biological basis for observed huanglongbing tolerance in some existing cultivars, and will greatly aid in the development of more tolerant cultivars in the future. 05 Potential mitigation of huanglongbing through application of antibacterial compounds. Agriculture Research Service (ARS) researchers in Ft. Pierce, Florida, led a cooperative research and development agreement project assessing potential mitigation of huanglongbing through application of antibacterial compounds. Data collected showed statistically significant improvement in tree health and pathogen titer reduction in most field plots, and some showed improved cropping. The need for improved citrus cropping is so great that these data provided support for a �Crisis Declaration of Use� by the Florida Ag Commissioner and the associated antibacterials have been applied to a majority of Florida citrus acreage. Enhanced productivity in existing citrus groves is needed to sustain the citrus industry and permit implementation of long-term solutions.
Impacts
(N/A)
Publications
- Hao, G., Pitino, M., Duan, Y., Stover, E. 2016. Reduced susceptibility to Xanthomonas citri in transgenic citrus expressing the FLS2 receptor from Nicotiana benthamiana. Molecular Plant-Microbe Interactions. 29:132-142.
- Albrigo, G., Stover, E.W. 2015. Effect of PGRs and fungicides on huanglongbing-related preharvest fruit drop of citrus. HortTechnology. 25:785-790.
- De Oliveira, M.L.P, Moore, G., Thomson, J.G., Stover, E.W. 2015. Agrobacterium-mediated transformation of Mexican lime (Citrus aurantifolia Swingle) using optimized systems for epicotyls and cotelydons. Advances in Bioscience and Biotechnology. 6:657-668.
- Bowman, K.D., McCollum, T.G., Albrecht, U. 2016. Performance of 'Valencia' orange (Citrus sinensis [L.] Osbeck) on 17 rootstocks in a trial severely affected by huanglongbing. Scientia Horticulturae. 201:355-361.
- Hall, D.G., Albrecht, U., Bowman, K.D. 2016. Transmission rates of �Ca. Liberibacter asiaticus� by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. Journal of Economic Entomology. 109:558-563.
- Bowman, K.D., Mccollum, T.G. 2015. Five new citrus rootstocks with improved tolerance to huanglongbing. HortScience. 50:1731-1734.
- Bowman, K.D., Albrecht, U. 2015. Comparison of gene expression changes in susceptible, tolerant, and resistant hosts in response to infection with citrus tristeza virus and huanglongbing. Journal of Citrus Pathology. 30450.
- Albrecht, U., Fiehn, O., Bowman, K.D. 2016. Metabolic variations in different citrus rootstock cultivars associated with different responses to Huanglongbing. Plant Physiology and Biochemistry. 107:33-44.
- De Oliveira, M.L., Thomson, J.G., Stover, E.W. 2016. High-efficiency propagation of mature 'Washington Navel' orange and juvenile "Carrizo" citrange using axillary shoot proliferation. HortTechnology. 26:278-286.
- Stover, E., Inch, S., Richardson, M., Hall, D.G. 2016. Conventional citrus of some scion/rootstock combinations show field tolerance under severe huanglongbing disease pressure. HortScience. 51:127-132.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. This is the second 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. The current project also initiates new work to address current threats and problems in U.S. citrus production. Hybridizations for improved citrus scion production were conducted using 68 parental combinations and about 1400 flowers in 2015. About three thousand new hybrid seedlings were produced from 2014 crosses for scion and rootstock development. Greenhouse studies continued to assess rootstock and scion tolerance of Candidatus Liberibacter asiaticus infection and huanglongbing disease. Greenhouse and field studies continued to indicate significant differences among plant genotypes with respect to tree tolerance or resistance to huanglongbing, the disease caused by Candidatus Liberibacter asiaticus. Influence of scion genotype on tolerance to huanglongbing appeared of a larger magnitude, but rootstock influence on tolerance was also significant. Work also continued to assess rootstock tolerance to Citrus tristeza virus and calcareous soils. Data on fruit crop, tree size, and health were collected from more than ten established rootstock and scion field trials. Three new rootstock field trials were planted. Trees were prepared for planting of four additional field trials with new hybrid rootstocks later in 2015. About 14,000 new propagations of new hybrid rootstocks were completed to prepare trees for budding and planting in additional field trials in 2016. Cooperative work continued with three commercial nurseries to multiply promising hybrid rootstocks to produce trees for medium-scale commercial plantings. Cooperative work was initiated under external funding with a university partner to establish twelve new rootstock field trials in Florida with the most promising rootstocks for huanglongbing tolerance. Progress has been made to characterize apparent tolerance to huanglongbing in conventional citrus suitable for use as scions, including about 4,000 that are sweet orange-like. Collaborations continue to show that the aroma volatiles in many of the advanced sweet-orange- like selections are very similar to true sweet orange, making it likely they can be officially designated as �sweet orange� for commercial use. Several of these selections appear to be much more resistant to huanglongbing (HLB) than are true sweet oranges. Initiated replicated commercial plantings of our most advanced sweet orange-like hybrids and mandarins alongside standards. Grant funded studies continued to produce and test transgenic rootstocks and scions, targeting increased tolerance to huanglongbing and canker diseases. More than five hundred new transgenic rootstocks and scions were produced. Transformation of scions included work with single chain fragment variable antibodies that bind to specific regions of huanglongbing pathogen proteins. Genes expressing 18 different single chain fragment variable antibodies were used to create hundreds of transgenic citrus plants. We will test the hypothesis that these proteins can interfere with the activity of these proteins in transgenic citrus and thereby protect them from colonization by the bacteria that causes huanglongbing. Transformation of new rootstocks focused on manipulation of the citrus resistance genes CtNDR1, CtMPK4, CtTGA7, CtNHL125, CtFAD7, CtNHO1, CtMIR2, and CtOSM34. Testing of selections for Liberibacter tolerance was conducted by controlled inoculation in the greenhouse using the vector Asian citrus psyllid. Three separate inoculation tests were conducted, involving nearly one thousand plants and more than twenty thousand caged psyllids. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with Liberibacter. Several transgenic rootstock selections showing increased resistance to Liberibacter have been identified from groups transformed with other resistance genes, and are also being prepared for further testing. Demonstrated reduced susceptibility to citrus canker in transgenic citrus expressing the FLS2 receptor from Nicotiana benthamiana. Transgenic citrus expressing a modified citrus protein and a chimera of that protein show substantially reduced susceptibility to citrus canker, and in preliminary studies, appear remarkably tolerant to huanglongbing. Focused work continued with twelve new promising rootstock hybrids that were identified as tolerant to huanglongbing disease by superior performance in replicated field trials. Five new huanglongbing (HLB) tolerant rootstocks were released for commercial use in Florida and plant material is being distributed through the Florida agency that regulates distribution of citrus within the state. Proliferating in-vitro shoot cultures (stage 2 cultures) were established from juvenile and mature explants of two rootstock selections, Carrizo and US-942, to be used for transformation, germplasm conservation, and micropropagation. We are currently multiplying these cultures to determine their use in genetic transformation of citrus. Accomplishments 01 Released five new citrus rootstocks with tolerance to huanglongbing disease. ARS researchers at Ft. Pierce, Florida identified and released five new citrus rootstocks that display much higher sweet orange fruit productivity and tree health in multiyear field trials growing in the Florida east coast area severely affected by huanglongbing, as compared with commercial standard rootstocks. The new rootstocks, US-1279, US-1281, US-1282, US-1283, US-1284, are available from the Florida clean budwood program, and are being propagated for entry into large scale grower trials in 2015. The results from field testing indicate that these rootstocks enable sweet orange trees to better tolerate huanglongbing disease. Trees on these rootstocks become infected with the disease, but after infection these trees yield more fruit that have higher sugar content in comparison to orange trees on other standard rootstocks. The results suggest that use of these or other tolerant rootstocks will be a key component of successful citrus production management in the presence of huanglongbing disease. 02 Developed ARS-MEDIA for calculating media recipes based on ion-specific constraints. ARS researchers at Ft. Pierce, Florida developed ARS- MEDIA, an ion solution calculator that uses Microsoft Excel to generate recipes of salts for complex ion mixtures specified by the user. The spreadsheet is based on an algorithm published, by the author, in Nature Methods. The spreadsheet was made available to the public via the ARS software website (http://www.ars.usda.gov/services/software/ software.htm). The software is required for any experiment designed to determine ion specific effects. One major use of the software is in developing improved fertilizer compositions and mineral nutrient formulations for use in various agricultural applications � plant tissue culture, hydroponic, greenhouse & field fertilizer studies, and algal growth. Fields where ion-specific effects are important include agriculture, biology, ecology & environmental science, medicine, animal science, chemistry, and physics. 03 Developed transgenic citrus with reduced susceptibility citrus canker disease. Transgenic citrus plants were produced expressing the FLS2 receptor from Nicotiana benthamiana. These transgenic plants show substantially reduced susceptibility to citrus canker, and increased tolerance to huanglongbing disease. These disease resistant or tolerant cultivars are of great interest because of the large commercial importance of these two diseases in the U.S. citrus industry.
Impacts
(N/A)
Publications
- Furr, J., Reece, P., Kahn, T., Siebert, T., Barry, G., McCollum, G., Castle, W., Stover, E. 2014. �US Furr� and �US Furr-ST� mandarin. Journal of American Pomological Society. 68:198-203
- Bai, J., Baldwin, E.A., Hearn, J., Driggers, R.E., Stover, E.W. 2014. Volatile Profile Comparison of USDA Sweet Orange-like Hybrids versus �Hamlin� and �Ambersweet�. HortScience. 49(10):1262-1267.
- Stover, E.W., Mccollum, T.G., Ramos, J.E., Shatters, R.G. 2015. Growth, health and Liberibacter asiaticus titer in diverse citrus scions on mandarin vs. trifoliate hybrid rootstocks in a field planting with severe huanglongbing. Proceedings of Florida State Horticultural Society. 127:53- 59.
- Stover, E.W., McCollum, T.G., Driggers, R.E., Lee, R.F., Shatters, R.G., Duan, Y.P, Ritenour, M.A., Chaparro, J., Hall, D.G. 2015. Resistance and tolerance to huanglongbing in citrus. Acta Horticulturae. 1065:899-903.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. This is the first year of a new project. The new project continues the long-term goals of a previous project (6618-21000-013-00D) to develop new citrus scion and rootstock cultivars with traits critical for successful commercial production and marketability. The new project also initiates new work to address current threats and problems in U.S. citrus production. Data on fruit crop, tree size, and health was collected from more than twenty established rootstock and scion field trials. Five new rootstock field trials were planted. Over three thousand new hybrid seedlings were field planted for evaluation of scion traits. Trees were prepared for planting of three additional field trials with new hybrid rootstocks later in 2014. About twelve thousand new propagations of new hybrid rootstocks were completed to prepare trees for budding and planting in additional field trials in 2015. Work began to construct an additional greenhouse to propagate hybrid rootstocks for field trials. Cooperative work continued with three commercial nurseries to multiply promising hybrid rootstocks to produce trees for medium-scale commercial plantings, and an agreement is being set up with a fourth commercial nursery to begin similar work. Greenhouse studies continued to assess hybrid rootstock tolerance of Candidatus Liberibacter asiaticus infection and huanglongbing disease. Work also continued to assess rootstock tolerance to Citrus tristeza virus, calcareous soils, and salinity. Progress has been made to characterize apparent tolerance to Liberibacter-huanglongbing in conventional citrus. A citrus hybrid with commercial-quality fruit has grown extremely well compared to standards in a replicated trial under heavy Liberibacter-huanglongbing pressure. Trees of this new hybrid are large and are visibly fully healthy and cropping heavily despite Liberibacter infection. In contrast, sweet orange trees are much smaller, have substantial dieback, and no fruit. Grant funded studies continued on defense-related genes and small RNAs associated with Liberibacter-huanglongbing, in collaboration with University of Maryland and University of California research groups. A study of localized defense gene expression in shoots and roots provided evidence of striking differences that are a major advance in understanding, and yield strong insights into ways to overcome the disease. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility provides additional insights into disease progression and the potential for improved management. During the year, more than 2000 seedlings were produced from 2013 crosses to develop improved citrus scions or rootstocks, and are being grown out for evaluation. In 2014, crosses were made with 48 parental combinations, emphasizing genetic seedlessness for scions, and Liberibacter-huanglongbing tolerance for scions and rootstocks. Three hundred to five hundred buds for each of eight advanced scion selections have been irradiated to generate low-seeded or better-colored variants. Seven new quality scion selections were identified and provided to a collaborator to be shoot-tip grafted for disease clean-up. Ten new promising rootstock hybrids were identified by superior performance in replicated field trials, and clean material was prepared for release and commercial distribution. Cooperative projects were established with commercial nurseries to begin propagation of the most promising new rootstocks. Factors potentially important for efficient genetic transformation of juvenile and mature citrus tissue were investigated and included types, proportions and concentrations of plant growth regulators, mineral nutrients, carbon sources, explant sources, preconditioning treatments, culture matrix, and genotype. Experiments to develop proliferating in vitro shoot cultures have been initiated for juvenile Carrizo citrange and Citrus macrophylla. Nutritional and plant growth regulator effects are being determined in these experiments. As conditions are identified that result in vigorous stage 2 cultures, mature tissue and a broader range of types will be examined. Accomplishments 01 New sweet orange-like hybrid with tolerance to huanglongbing (HLB) disease. ARS researchers at Ft. Pierce, Florida have developed a new hybrid with high-quality sweet orange-like fruit that displays excellent tolerance to HLB disease. The selection is a hybrid between citrus and trifoliate orange, but unlike other similar hybrids, has commercial quality fruit which is comparable with sweet orange in flavor. The selection has grown extremely well compared to standards in a replicated trial under heavy HLB pressure. Trees have been propagated at a commercial nursery and will be placed in replicated plantings on six grower sites with other advanced sweet-orange-like selections and industry standards in 2015. 02 Nine new citrus rootstocks with tolerance to huanglongbing (HLB) disease. ARS researchers at Ft. Pierce, Florida have identified nine new citrus rootstocks that display much higher sweet orange fruit productivity and tree health in field trials growing in areas severely affected by HLB, as compared with commercial standard rootstocks. These rootstocks, US-1279, US-1281, US-1282, US-1283, US-1284, US-1293, US- 1317, US-1319, and US-1321 have been entered into the Florida clean budwood program, and are being propagated for entry into large scale grower trials in 2015. The results from field testing indicate that these rootstocks enable sweet orange trees to better tolerate huanglongbing disease, and suggest that use of a tolerant rootstock will be one component of successful citrus production management in the presence of that disease. 03 Developed a liquid culture method suitable for genetic transformation. Citrus types vary widely in their transformability on agar media, with scion types such as sweet orange and grapefruit having low transformation efficiencies, often much less than one percent. Improving transformation of the scion types would greatly reduce the resources required to widely utilize transgenic technology in citrus and may be facilitated by using a liquid culture system. ARS researchers at Ft. Pierce, Florida examined numerous factors and determined their effects on shoot regeneration and/or genetic transformation efficiency. A liquid culture system was developed and found to be suitable for shoot regeneration and genetic transformation. The liquid system resulted in in vitro responses that were comparable to agar, and in some cases some improvement was observed. For example, genetic transformation efficiency was generally higher in the liquid system, possibly due to fewer escapes.
Impacts
(N/A)
Publications
- Albrecht, U., Hall, D.G., Bowman, K.D. 2014. Transmission efficiency of Candidatus Liberibacter asiaticus and progression of Huanglongbing disease in graft and psyllid-inoculated citrus. HortScience. 49(3):367-377.
- Stover, E.W., Richardson, M.L., Driggers, R., Hall, D.G., Duan, Y.P., Lee, R.F. 2014. Incidence and severity of Asiatic citrus canker on citrus and citrus�related germplasm in a Florida field planting. HortScience. 49:4-9.
- Lu, H., Zhang, C., Albrecht, U., Shimizu, R., Wang, G., Bowman, K.D. 2013. Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis. Frontiers in Plant Science. 4(157):1-10.
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