This is a 3-year project with 3 main focal points: 1. To build a foundation of understanding of the host- Candidatus Liberibacter asiaticus (LAS) interactions that involves testing multiple genetic variants of citrus, examination of environmental and seasonal effects on symptoms and pathogen replication and movement, and understanding effects of genetic variation in the pathogen system, so that better methods of controlling the vector, detecting and monitoring the disease, and growing trees will allow better production of citrus; and, 2. To develop an understanding of how Las interacts with citrus genotypes to cause disease in sensitive varieties and to not cause disease in tolerant varieties in the hope that this information will lead to the development of approaches to produce citrus economically in the present situation with citrus greening in Florida; and, 3. To provide knowledge and resources to support and foster research in other laboratories. A substantial number of projects to be supported are based on our research and reagents (propagated healthy and HLB-infected plants, HLB inocula, nucleic acid extracts, etc.) supplied by our laboratory. We view this as one of our most important goals. Year 1 milestones: 1) Propagate and prepare trees for year 2. We have received seed from a range of different citrus relatives from the National Citrus Repository in Riverside, CA, and these plants are growing to be tested for response to HLB. 2) Begin screening of commercial varieties and additional citrus relatives to Las. We have an experiment underway to test a range of different sweet orange and grapefruit varieties for sensitivity to HLB. The experiment also has some citrus relatives. HLB symptoms have just started to develop. 3) Obtain seeds fo Poncitrus trifoliata and poncirus hybrids from a series of independent sources, propagate plants, and start experiments to evaluate phenotypic differences in their response to HLB. We have several independent selections of Poncirus trifoliata growing. We also have a series of experiments underway to analyze Poncirus rootstocks and interstocks on the development of disease. 4) Evaluate C. latipes response: repeat an attempt to initiate infection of C. latipes trees using different sources of the trees, on different rootstocks, and using different sources of HLB inoculum. We have a series of C. latipes trees infected with a series of different Las isolates, but nothing has induced the original symptoms that were associated with non-detection of Las. However, we have an interesting observation that the original trees are greatly inhibited when used as budwood. We are investigating this phenomenon. 5) Begin microscopy studies and develop an approach to analyze sugar metabolism in sensitive vs tolerant hosts in an attempt to understand what determines a differential host response to HLB. We have found that phloem necrosis in stems parallel the degree of disease severity. Citrus that is tolerant to HLB has much less phloem necrosis than sweet orange and grapefruit.
This is a continuing project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. With the loss of trees due to canker eradication and development and the continuing losses to greening, there is a major concern whether sufficient fruit will be produced to keep processing plants open. We are approaching this problem in two ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen to focus on antibacterial peptides because they represent one of the few choices available for this time frame. Secondly, we are developing virus vectors based on CTV to express the antibacterial peptides in trees in the field as an interim measure until transgenic trees are available. We think that this approach could be used beginning 2-3 years from now and until probably 15 years from now when resistant trees should be available. The milestones for this year’s research are: 1) Continue to screen peptides for activity against Las in citrus trees using the CTV vector. We now have built 45 different peptide constructs that are being tested against HLB or citrus canker. We have 5-7 peptides that we think have activity against these bacteria. We also are developing a more natural screening assay using psyllids to infect trees with HLB. 2) Begin high-throughput screening in tobacco against solanaceous liberbacter. (Falk, UC Davis) The subcontract to UC Davis has not been completed yet. However, Dr. Falk has established colonies of the solanaceous psyllid. 3) Improve CTV-based vector to produce 2-5 peptides and to overcome cross-protection. We have built several new versions of CTV vector and have shown that two foreign genes can be expressed efficiently at the same time. We are making progress in understanding the process of cross protection to allow development of a CTV vector that will allow subsequent application of antibacterial gene products. 4) Examine survival of peptides in fruit and juice. We have developed the plants for these assays, but this project is just beginning. 5) Prepare trees for year 2. As we develop new anti-bacterial or anti-psyllid constructs into the CTV vector, they immediately begin their pathway to citrus trees to be tested for resistance or tolerance to HLB or citrus canker.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. We proposed to expand our current research to different citrus varieties which are either susceptible or tolerant to HLB and a more facile model system tobacco (Nicotiana tabacum Xanthi). The specific objectives of this proposal are: transcriptional and microscopic analyses of citrus varieties which are either susceptible or tolerant to Ca. L. asiaticus infection at different infection stages in greenhouse and citrus grove; and transcriptional and microscopic analyses of host response to Ca. L. asiaticus infection with a model system tobacco. Microarray analysis and/or suppressive subtractive hybridization libraries approaches will be used to study the host response to the HLB pathogen infection followed by confirmation with Northern blot or quantitative reverse transcriptional PCR. Anatomical study will be performed with light microscopy using different staining methods. The following objectives related to this project were pursued: (A) An initial microarray analysis of host response of sweet orange to Las infection in greenhouse and in field; and (B) A preliminary study of the effects of HLB on phloem at the microscopic level and phloem transport of sweet orange. The results of these experiments are detailed below. Investigation of the host response was examined with citrus microarray hybridization based on 33,879 expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The expression of a total of 624 genes was significantly changed: 307 genes were up-regulated and 317 genes down-regulated in inoculated trees. The host genes that showed changes in expression pattern induced by Las infection were related to plant pathogenesis/stress (10.4% of the total), anthocyanin biosynthesis (0.5%), cell wall metabolism (6.6%), cell division (0.96%), detoxification (6.2%), lipid metabolism (2.4%), metabolite transport (5.8%), metal transport (2.6%), nucleotide metabolism (1.6%), phenylpropanoid/flavonoid/terpenoid metabolism (5.6%), phytohormones (3.0%), protein kinase (5.0%), protein metabolism (3.8%), protein-protein interaction (1.1%), signal transduction (1.8%), sugar metabolism (4.3%), transcription/translation factors (7.2%) and unknown/hypothetical genes (31.1%). The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since ÔCa. Liberibacter asiaticusÕ does not form aggregate in citrus. The upregulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants. To further expand our current understanding of Las-host interaction, we are currently comparing two susceptible and two resistant hybrid/cultivars. Currently, we are collecting samples for more than 4 months. The gene expression and anatomical study are under way.
Citrus Huanglongbing (HLB) is one of the most destructive diseases on citrus. Very little is known about the causal pathogen and the microbiome associated with the infected trees. The goal of the proposed research is to characterize the bacteria (endophytes and plant pathogens) associated with HLB positive citrus, the Asian citrus psyllid, dodder, and periwinkle. The specific objectives of this proposal are: (1) Comparison of the microbiomes in leaf midribs and roots of HLB pathogen positive and free citrus of different varieties; (2) Characterization of the microbiomes that can be transferred among citrus, psyllid, dodder, and periwinkle; and (3) Understanding how Candidatus Liberibacter asiaticus colonizes and moves inside the phloem. Comparison of the microbiomes associated with HLB pathogen positive and negative citrus will illuminate the causal agent of citrus greening. Potential beneficial endophytic microorganisms could be identified from escape plants which survived in heavily infected citrus groove with HLB. Beneficial microorganisms have been shown in previous studies to have the capacity to control plant diseases by accelerating seedling emergence, promoting plant growth and development, and preventing the invasion of plant pathogens. The investigation of the microbiomes associated with different hosts will help understand the transmission of microorganisms between different hosts. Major achievements: Candidatus Liberibacter asiaticus (Las) as the pathogen responsible for HLB disease in Florida. Our data indicates that Ca. Liberibacter asiaticus is the pathogen responsible for HLB disease in Florida. The bacterial diversity associated with citrus leaf midribs was characterized for citrus groves that contained the HLB pathogen. We employed a combination of high-density phylogenetic 16S rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for symptomatic and asymptomatic citrus midribs. PhyloChip analysis indicated that 47 orders of bacteria in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. Only Las was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of Las in symptomatic leaves. On average, more than 86% of the clones from symptomatic trees were Las. These data implicate Las as the pathogen responsible for HLB disease in Florida. Isolation of plant growth promoting bacteria from potential escape citrus Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways controlling the HLB disease. Fifty-four morphologically distinct isolates were obtained from surface sterilized roots of symptomatic and asymptomatic (potential escape trees) citrus plants from a citrus grove with a HLB infection rate of more than 60% and an infection history of approximate five years. We used a detailed approach for screening novel isolates by conducting qualitative and quantitative assays for traits related to mineral nutrition (phosphate solubilization, siderophore production), development (phytohormone synthesis), health (production of antibiotics), induction of systemic resistance (salicylic acid production), and stress relieve (production of 1-amino-cyclopropane-1-carboxylate deaminase). Qualitative screening showed that for all of these criteria, asymptomatic plants harbor a significant greater diversity of potentially beneficial bacterial strains. In vitro quantitative assays showed that the isolates from the asymptomatic trees have significantl greater beneficial abilities.
Citrus Huanglongbing (HLB) is the biggest threat to the Florida citrus industry. Florida accounted for 70% of the total U.S. production in 2006-07. HLB, known as citrus greening, can debilitate the productive capacity of citrus trees with losses of 30-100% reported. HLB was found in Florida in 2005 and has spread throughout thirty of the citrus producing counties in Florida by January 2008. The overall goal of this proposed work is to characterize the virulence mechanisms of Candidatus Liberibacter asiaticus (Las), the citrus Huanglongbing (HLB) pathogen, thus to come up with new management strategies by genome sequencing and functional genomics approaches. The original goal of the proposed research is to further complete the genome sequencing of Candidatus Liberibacter asiaticus, for which a draft sequence is available. The goal was modified to meet the current progress in genome sequencing of Candidatus Liberibacter asiaticus with the advice and permission from program manager of FCPRAC. The tile has been changed to the following to better suit the goal: Understand the virulence mechanism of Candidatus Liberibacter asiaticus by genome sequencing and functional genomics approaches. Bioinformatics analysis was performed to identify potential virulence factors. The SignalP v3.0 program was used to predict the presence of signal peptide within the proteins. The secretomeP 2.0 program was used to predict the non-classical secretion proteins without signal peptide. ORF containing transmembrane domains was predicted by TMHMM2.0 program. Smart and other programs were used to further mine the genome sequence of Candidatus Liberibacter asiaticus. Progress was made on characterization of the virulence mechanism of Candidatus Liberibacter asiaticus. Both microarray and real time PCR approaches were used to compare the gene expression profile of Candidatus Liberibacter in planta and in psyllids. The microarray covers all the genes of Candidatus Liberibacter asiaticus. Genome of the HLB pathogen was analyzed for homologs of known virulence genes, genes containing domains and motifs which may play roles in virulence and pathogenicity. Totally, 560 genes were included in quantitative revere transcriptional PCR analysis. It is expected that genes which are critical for survival and growth in planta and psyllids, virulence in citrus, transmission in psyllids will be differentially expressed in planta and in psyllids. Totally, more than 260 genes were identified which are differentially expressed in planta and in psyllids. Among them, some genes were shown to be potential virulence factors. Our preliminary results suggest that Candidatus Liberibacter asiaticus affecting gene expression of the host by secreting some putative virulence factors into the phloem of citrus. Further functional analysis is of those potential virulence factors is being performed in the Wang lab and collaborators lab. For example, both gene ontology and KEGG pathway analyses are being performed to further understand the transcriptional profiling of the HLB pathogen in different environments. Analysis of the genome of the HLB pathogen also revealed multiple potential target of developing bactericides and other approaches of controlling HLB. The potential of utilizing those approaches are being tested.
Objectives: 1. Establish the proper protocol in terms of sampling technique, iodine solution, and parameters to be used for visual detection. 2. Analyze starch accumulation patterns in symptomatic and asymptomatic leaves from trees infected with Citrus greening. 3. Determine any limitations based on varietal differences. 4. Distinguish between greening and other biological diseases and horticultural deficiencies in terms of starch staining. After a year of experimentation, we accomplished all four original goals listed above. 1. Establish the proper protocol in terms of sampling technique, iodine solution, and parameters to be used for visual detection. A protocol was established that maximizes the visualization of leaf starch accumulated as result of HLB infection. The method, based on iodine staining, was tested and appears to be equally effective in all varieties and cultivars investigated. Published by IFAS EDIS publications, the test is now widely used to detect suspicious HLB trees. 2. Analyze starch accumulation patterns in symptomatic and asymptomatic leaves from trees infected with HLB. For the most part, there was a direct correlation between yellow HLB symptom patterns and starch accumulation. This rule seems rather universal and fails only in the leaf pattern known as ‘green islands’, where starch seems to accumulate in random pockets. Asymptomatic leaves collected from branches already containing symptomatic leaves usually show a high degree of starch accumulation. Sometimes, the amounts of starch are visually indistinguishable from symptomatic leaves in the same branch. On occasion, there are asymptomatic and starch-less leaves mixed within symptomatic starch-laden leaves. 3. Determine any limitations based on varietal differences. From the extensive studies conducted throughout one year, we detected 2 circumstances where the starch test may not be as accurate. One, Murcott oranges seem to have a natural high base level of starch that makes the process of decision making at low and intermediate levels more difficult. Second, acid limes do not appear to follow a consistent pattern of starch accumulation in leaves. Although the numbers of samples has been small, we can not at this time recommend the iodine test on acid limes or lemons. 4. Distinguish between greening and other biological diseases and horticultural deficiencies. The only cultural practice with potential effect on starch accumulation is Zn deficiency-induced starch accumulation, albeit lower levels. These low levels are comparable to those found in Murcott. However, since Zn deficient presents characteristically different symptoms from HLB, this should not present a problem. No other disease common to Florida citrus resulted in starch accumulation.
Significant progress has been made in the identification, shoot-tip grafting (STG)ing, grafting in the greenhouse, testing, increase and release of cultivars needing STGing. Over 111 Citrus clones are currently in the STGing program. The last three remaining Florida Citrus Arboretum selections were set up as budsticks for the purpose of heat therapy and obtaining shoots for STGing. During this first quarter over 633 STGs were set up. These represented 18 varieties including 7 rootstocks, 4 breederÕs selections, two Florida Citrus Arboretum selections, 4 difficult varieties and one field rescue. During the same time period, 41 successful STGs were grafted onto rootstocks in the greenhouse. These represented 15 varieties which included 7 rootstocks, 4 breederÕs selections, 3 difficult varieties and one field rescue. Over 140 real-time PCR tests were performed on STGs that grew to sufficient size in the green house. To allow clean budwood to be propagated as soon as possible, 167 increase trees were propagated from tested original STGs. Twenty varieties completed testing and were released; 5 were breeder selections and the remainder was mainly STGed to remove Citrus tristeza virus (CTV). At the remote and secure Citrus Budwood Foundation in Chiefland, 18 fully tested and released STGs or their propagations were planted. These were either new to Chiefland or replaced varieties that hitherto were only available with mild isolates of CTV. Conventional Citrus tatter leaf virus PCR has been converted to a SYBR green real-time PCR assay as a first step. The initial attempt to create a Taqman assay failed due to the sequence variability of the isolates. Another attempt will be made. The equipment is essential for the success of this grant. The money is almost in place and quotes for requisitions are ready to go; the equipment should be in place by the next quarterly report. The number of candidates to be shoot-tip grafted is still over one hundred as 10 new parent candidates were entered for STGing during this quarter.
Valencia fruit were harvested and juiced, with blends of symptomatic greening fruit (based on % weight of fruit) as previously described for Hamlin fruit. Blends include 0% greening (negative control), 2.5% greening, 5% greening, 10% greening, 20% greening, 50% greening, and 100% greening (positive control). Due to breakdown of the microthermics juice pasteurizer, all juice remains at -20’C until equipment is repaired. We evaluated impacts of HLB and girdling on oil compounds in ‘Valencia’. The girdle treatments were performed on trees as previously reported. Fruit affected by HLB and/or girdling were collected in April 2011. At least 8 trees for each treatment, and at least 4 fresh fruit from each tree (a biological replicate) were used for this study. The collected samples include healthy (H), asymptomatic (AS), symptomatic (SA), ungirdled (UG), half girdled (HG), and full girdled (FG) fruit. The cold-water press method was performed to extract total oil from fresh fruit flavedo. Results indicated H, AS, UG, and HG fruit had similar amount of oils (from 5.5 to 6.1 .l of oil/g fruit weight). Oil content was significantly reduced in SA and FG fruit (3.0 and 3.2 .l of oil/g fruit weight). GC-MS analysis was performed to compare the oil components in all of the treatments. Uneven pigmentation was observed in HLB and FG fruit peel. Thus, oil for GC-MS analysis was extracted from stem end, equator and blossom end separately to account for positional differences, if any. Here we report on oil extracted from the fruit equator. For each replicate, 0.1.l oil was directly injected into GC-MS. 37 compounds were identified by GC-MS analysis. Principal component analysis (PCA) will be performed to statistically analyze data from all treatments from different fruit positions when completed. When compared with UG fruit, the accumulation of 9 out of 37 volatiles was changed in FG, including octanal (0.2x), linalool (0.1x), trans-P-mentha-2 (0.5x), limonene oxide (3x), citronellal (53x), .-terpineol (0.4x), decanal (0.3x), neryl acetate (2.6x) and dodecanal (0.4x). Only one volatile (citronellal) was found altered in HG compared to UG fruit. When compared with H fruit, 10 compounds were shifted in SY fruit, including octanal (2x), .-terpinolene (0.5x), nonanal (0.4x), limonene oxide (2x), citronellal (41x), .-bergamotene (0.5x), dodecanal (0.5x), .-cubabene (0.5x), .-farnesene (3.1x) and .-sinensal (3.3x). Five volatiles were also changed in AS fruit when compared with H fruit. The preliminary results indicate that HLB and girdling altered the amount and components of citrus oil. Accumulation of 7 volatiles was affected by HLB but not girdling. Those include octanal, .-terpinolene, nonanal, .-bergamotene, .-cubabene, .-farnesene and .-sinensal.
Valencia fruit were harvested and juiced, with blends of symptomatic greening fruit (based on % weight of fruit) as previously described for Hamlin fruit. Blends include 0% greening (negative control), 2.5% greening, 5% greening, 10% greening, 20% greening, 50% greening, and 100% greening (positive control). Due to breakdown of the microthermics juice pasteurizer, all juice remains at -20’C until equipment is repaired. We evaluated impacts of HLB and girdling on oil compounds in ‘Valencia’. The girdle treatments were performed on trees as previously reported. Fruit affected by HLB and/or girdling were collected in April 2011. At least 8 trees for each treatment, and at least 4 fresh fruit from each tree (a biological replicate) were used for this study. The collected samples include healthy (H), asymptomatic (AS), symptomatic (SA), ungirdled (UG), half girdled (HG), and full girdled (FG) fruit. The cold-water press method was performed to extract total oil from fresh fruit flavedo. Results indicated H, AS, UG, and HG fruit had similar amount of oils (from 5.5 to 6.1 .l of oil/g fruit weight). Oil content was significantly reduced in SA and FG fruit (3.0 and 3.2 .l of oil/g fruit weight). GC-MS analysis was performed to compare the oil components in all of the treatments. Uneven pigmentation was observed in HLB and FG fruit peel. Thus, oil for GC-MS analysis was extracted from stem end, equator and blossom end separately to account for positional differences, if any. Here we report on oil extracted from the fruit equator. For each replicate, 0.1.l oil was directly injected into GC-MS. 37 compounds were identified by GC-MS analysis. Principal component analysis (PCA) will be performed to statistically analyze data from all treatments from different fruit positions when completed. When compared with UG fruit, the accumulation of 9 out of 37 volatiles was changed in FG, including octanal (0.2x), linalool (0.1x), trans-P-mentha-2 (0.5x), limonene oxide (3x), citronellal (53x), .-terpineol (0.4x), decanal (0.3x), neryl acetate (2.6x) and dodecanal (0.4x). Only one volatile (citronellal) was found altered in HG compared to UG fruit. When compared with H fruit, 10 compounds were shifted in SY fruit, including octanal (2x), .-terpinolene (0.5x), nonanal (0.4x), limonene oxide (2x), citronellal (41x), .-bergamotene (0.5x), dodecanal (0.5x), .-cubabene (0.5x), .-farnesene (3.1x) and .-sinensal (3.3x). Five volatiles were also changed in AS fruit when compared with H fruit. The preliminary results indicate that HLB and girdling altered the amount and components of citrus oil. Accumulation of 7 volatiles was affected by HLB but not girdling. Those include octanal, .-terpinolene, nonanal, .-bergamotene, .-cubabene, .-farnesene and .-sinensal.
Our study with Citrus sinensis ‘Valencia’ trees indicated fruit drop in HLB-infected trees was initiated at breaker stage. When compared with fruit in healthy trees,10-fold higher symptomatic fruit dropped to the ground and the detachment force of attached fruit (FDF) was reduced 75%. There was no difference of % abscission and FDF between asymptomatic fruit and healthy fruit. When compared with abscission zones of healthy fruit, the expression of abscission-induced genes such as 1-aminocyclopropane-1-carboxylate synthase (CsACS1), cellulose-a1 (CsCel-a1), polygalacturonase (CsPG), phospholipaseA2. (CssPLA2.) and PhospholipaseA2. (CsPLA2.) were 17-, 7-, 530-, 1.5-, and 2-fold higher in symptomatic fruit abscission zones respectively. The expression of CsACS1 and CsCel-a1 were 5-fold increased and CsPG was 150-fold increased in asymptomatic fruit abscission zones. In addition, ethylene production was 6- and 2-fold higher in the areas of symptomatic and asymptomatic fruit abscission zones respectively. HLB increased leaf abscission by 7% after 80 observation days. A 10% reduction of the detachment force and increased expression of abscission-induced genes were also found in symptomatic laminar and petiole abscission zones compared to healthy controls. We analyzed girdled tissues to study the impacts of carbohydrate deprivation on abscission and HLB-like symptom development. A bark full ring or half ring around the twig located 10-cm above a fruit was removed at immature stage. Leaves between the girdled region and fruit were removed. Girdling was also applied on a branch that had between 50 to 100 of leaves. The study showed symptomatic and full girdled fruit shared similar impacts on size, weight, and color. Lopsided symptoms were only found in symptomatic, but not girdled fruit. In most of the full girdled fruit, the flavedo and immediately underlying regions of albedo very close to fruit abscission zone was orange in color. Mottled chlorosis appeared on leaves subtending the full girdled areas. There was no visual differences between half-girdled compared to ungirdled tissues. Similar to symptomatic tissues, full girdled tissues showed the shift of starch and sucrose contents, premature fruit drop, leaf drop, and the increased expression of abscission-induced genes at abscission zones. However, the impacts of girdling were greater than HLB infection especially on tissue drop and carbohydrate metabolism.
During the final quarter of the year, the Core Citrus Transformation Facility (CCTF) maintained its level of performance and produced transgenic citrus plants for many satisfied customers. During the 4th quarter, the CCTF continued to utilize the provided funding to process the orders for transgenic plants, mostly servicing CRDF funded researchers studying transgenes with potential to generate resistance to HLB. Orders processed during this quarter include: pY46-Carrizo; pY102-Carrizo; pY141-Carrizo; pY150-Carrizo; pCitIntra-Duncan; pAZI-Duncan; pAtBI-Duncan; pBCR2-Duncan; pDPR1-Duncan; pLP1-Hamlin; pLP1-C-mac; pLP2-Hamlin; pLP2-C-mac. During the last quarter of this funding year, work was mostly concentrated on recent orders. Fourteen Duncan plants were produced carrying a gene of interest from the p35S-TRX vector and 23 more Duncan plants were produced carrying a gene from the pSucTRX vector. Multiple Duncan plants were produced toward satisfaction of ‘WG’ group of orders: eight-pWG22-1 plants, three-pWG21-1, and four pWG25-13 plant. Also, following Carrizo plants were produced for the ‘Yale’ order: nine plants with the gene from the pY46 vector and 11 plants with the gene from the pY102 vector. Eighteen Duncan plants were produced after treatment with bacteria harboring pBCR2 vector. Three more Duncan plants were produced with the EDS5 gene and six Mexican limes with the P35 gene. Four additional Duncan plants carrying a gene from pSUC-CitNPR1 were produced. The impact of citrus diseases on Florida citriculture is rapidly growing, and our participation in this battle is growing with some positive results already being published. Continued funding for CCTF which is an integral part of this community and contributes greatly towards common goal will allow for the progress to go on by keeping production of transgenic material un-interrupted and at high levels.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. A number of plant derived insecticidal transgenes, each driven by a root specific promoter were incorporated into Carrizo citrange. As part of this project we cloned four insecticidal genes and two plant derived promoters. Constructs were initially tested on N. benthamiana for confirmation of transgene activity. Carrizo citrange was subsequently transformed utilizing the conventional Agrobacterium mediated transformation process. A total of 123 putative transgenic lines were generated. PCR screening identified 74 of these lines to be transgenic. 54 of these transgenic lines had a high level of transgene expression as determined by qPCR. We have also identified a number of putative root specific genes from Citrus clementina by data mining the phytozome database. Of them several putative sequences were selected for characterization using qPCR. RNA was extracted from mature and juvenile roots from non-transgenic Swingle citrange. Each of the identified sequences were characterized in these different tissues. In addition, transcript levels in the leaves were also measured. The Cic1867m gene was determined to be very root specific and a 1.2 kb fragment of its promoter was cloned from Clementine genomic DNA using PCR. Deletion analyses identified a 0.8 kb fragment from that promoter fragment to be sufficient for root specific activity and transgenic plants were produced using this promoter. Cuttings from all the better performing lines have been made and are being rooted in the mist bed. These clones will be sized up for Diaprepes feeding experiments. Clonally propagated plants will be force fed with Diaprepes neonates – when available and root damage / insect mortality evaluated.
This project had a single objective to assure un-interrupted production of transgenic citrus plants through the services of Citrus Transformation Facility (CTF). Continued operation of the facility was going to guarantee a foundation regarding this aspect of research for the scientific community involved in fight against huanglongbing (HLB) and citrus canker. By producing transgenic plants and bringing into life the ideas of scientists working in different laboratories, CTF would facilitate the process of search for candidate plants that would be tolerant/resistant to diseases and provide major relief for Florida Citrus Industry. The objective of this project was successfully accomplished. Throughout the funding period of three and-a-half years, CTF operated without major interruptions and provided service in the form of production of transgenic plants. The level of production stayed at expected, satisfactory level and resulted in creation of 781 citrus plants that represent independent transgenic events. These plants belong to eight different cultivars: Duncan grapefruit, Carrizo citrange, Pineapple sweet orange, Mexican lime, Valencia sweet orange, Swingle citrumelo, Kumquat, and Pomelo.During this project, CTF worked on 103 orders that were placed by 10 clients and this group included eight faculty members based at University of Florida, one faculty from University of California and one Foundation. The number of projects for which the plants were produced was higher than the number of clients. For example, Dr. Nian Wang was one of the clients but orders from his lab were the part of at least five different projects done by his post-doctoral associates.All produced transgenic plants were associated only with research that has to do with disease resistance. This is a clear indication of the role CTF plays in the efforts to overcome effects of HLB and citrus canker.CTF supported the projects from Dr. Nian Wang’s lab that resulted in successful application of CRISPR/Cas9-mediated genome editing technology in Citrus. Plants produced this way will be the most market-friendly as they only have minor modifications of their own gene(s) and in the near future will be free of any other inserted DNA. The work done in CTF lead to production of Duncan grapefruit plants that exhibit high degree of resistance to citrus canker. Other commercially important cultivars could be made with improved resistance to canker based on results of this project.As a part of another research project aimed at control of Asian Citrus Psyllid (ACP) in citrus groves, transgenic Indian curry leaf plants were also produced in CTF. There are six such plants that are being tested for the ability to kill ACP after they were allowed to feed on their leaves. All these successes were achieved even though CTF experienced high flux of employees, had to move to temporary location for six months, and was exposed to adverse effects of hurricane Irma.Future opportunities for the CTF are determined by the needs of Florida Citrus Industry. As those needs change from the search for the solution to HLB and canker to a development of specialty fruit with consumer-oriented traits in post-HLB era, CTF will be there to support all these efforts.
There were three significant accomplishments during the quarter. A visit to the citrus sector of Sao Paulo, Brazil was completed in May. The trip included visits to two large citrus growing farms and several smaller farms. FUNDECITRUS, the major citrus research entity in Brazil was also visited and researchers there gave an update on the greening situation in Sao Paulo. They have developed a model of greening spread at the grove level which may prove valuable in grove level decision-making. A main finding from the trip is that greening is widespread in the central-east portion of the Sao Paulo citrus growing area. A large number of trees in this area have been eradicated and more eradication will occur in the near future. While new planting to replace eradicated trees was accomplished at a high rate in 2006-08, new planting has declined with lower fruit prices which will have implications for future fruit production. A second accomplishment is that a relationship with researchers at the University of Sao Paulo has been established which will foster information exchange. Dr. Margarete Boteon will visit Florida in August to make a presentation at the Southwest Florida Citrus Expo. She has recently published a study which was two case studies that analyzed the cost of production for citrus in Sao Paulo. This study provides detail on production practices and costs not published previously. It will be of great assistance to analyze both future production prospects and the competitiveness of the citrus sector in Sao Paulo. An update of the economic impact of the citrus sector on the economy of Florida was begun late in the quarter. With the loss of both tree numbers and acres planted across all citrus varieties grown in Florida, it is important to establish a new benchmark on the economic impact of the industry. A similar approach to previous studies is being taken in which an input-output model of the Florida economy is modified to identify the economic contribution of the citrus industry.
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