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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

2016 Annual Report


Objectives
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
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 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
1. 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.

2. 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.

3. 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.

4. 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.

5. 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.


None.


Review 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.