Understanding the transmission of CLas within the citrus tree remains one of the principal obstacles in the global efforts to undermine the pathogenicity of HLB (citrus greening). The movement of CLas has been assumed to follow the photoassimilate stream through the phloem. However, many observations based on our knowledge of the bacteria and general phloem anatomy have exposed inconsistencies with the accepted beliefs. The brevity of available information on the ultrastructural properties of citrus phloem sieve elements has hindered efforts to understand the spread of the disease within a tree. For example, lateral movement of CLas around an infected stem appears improbable given the size of cytoplasmic plasmodesmata connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from the roots to uninfected aerial tree parts through the phloem seems highly unlikely given the direction of phloem sap. To date we lack a thorough investigation into the ultrastructure of citrus phloem and the surrounding tissue, the potential pathways that CLas could utilize to move long distances through citrus trees, and the location of CLas habitat within different citrus tissue. Using a variety of grafting and girdling experiments, SEM, TEM, confocal, high resolution computed tomography, and PCR tissue analysis we aim to gain a better understanding of the anatomical traits that facilitate the spread of CLas through citrus. These data will allow us to develop new screening tools that breeders can use to select for resistant scion/rootstock combinations to confer resistance or tolerance to HLB. As of this progress report the Valencia/Swingle trees have had HLB+ tissue grafted onto them. Approximately 80% of the grafted tissue has produced new flush and we are waiting for the remainder to produce new tissue. The girdling experiments have been completed and we are actively monitoring the wounds and the healing process to make sure that the redifferentiated tissue does not act as a pathway for CLas. We have hired a part-time employee to begin learning the microscopy techniques so they are well positioned to start the anatomical analysis once the trees are ready. We expect to begin tree dissection within the next quarter, followed by another 6 months to allow the remaining trees to develop HLB symptoms.
This portion of the project investigated the effect of HLB on Hamlin and Valencia orange peel oil quality by analyzing the volatile and olfactory profiles. In one study, peel oil samples were evaluated by gas chromatography-olfactometry (GC-O), where a person sniffs the volatiles as they come off a GC column, by GC-mass spectroscopy (MS) for peak identification, and by a sensory descriptive panel evaluating oil on filter paper. Analysis by GC-O revealed 57 odor-active peaks of which 33 were identified by GC-MS, and 22 confirmed by smelling chemical standards by GC-O. All identified compounds have been preciously reported in citrus and orange essential oil. There were 7 aliphatic aldehydes (hexanal, octanal, nonanal, decanal, undecanal, decadienal, dodecanal), 4 monoterpene aldehydes (citronellal, neral, geranial, perilla aldehyde), 3 aliphatic alcohols (hexanol, octanol, nonanol), 7 monoterpene alcohols (sabinene hydrate, linalool, 4-terpineol, citronellol, nerol, geraniol, cis-carveol), 8 monoterpene hydrocarbons (.-pinene, sabinene, myrcene, .-phellandrene, .-3-carene, limonene, .-phellandrene, terpinolene), 8 sesquiterpene hydrocarbons (.-copaene, .-cubebene, .-caryophyllene, .-copaene, .-humulene, germacrene D, valencene, .-cadinene), 1 monoterpene oxide (limonene oxide), 1 acid (methyl-octanoate) and 1 monoterpene ketone (carvone). Furthermore, 24 unknown compounds were detected by smell. Paired comparisons looked at Hamlin healthy/Hamlin HLB, and Valencia healthy/Valencia HLB. Multivariate statistics (PCA) found no differences in peak intensities between Hamlin healthy and HLB samples. Likewise, panelists could not distinguish between healthy and HLB samples for Hamlin oil. For Valencia oil, LRI 1185, .-cadinene and LRI 1645 were only perceived in HLB samples. Other differences were for LRI 955, .-phellandrene, and terpinolene, which had higher intensities in Valencia healthy. For GC-MS analyses, 10 compounds were significantly different between Valencia healthy and HLB, but contrary to Hamlin oils, they presented higher peak areas in Valencia HLB than in Valencia healthy, except for hexanal and cis-p-mentha-2,8-dien-1-ol. No significant difference was perceived between healthy and HLB Valencia samples by panelists during the difference test. Likewise, there were no significant differences between Valencia healthy and Valencia HLB juice made from the same oranges as for the peel oil extract. Therefore, the small differences detected by GC-O between healthy and HLB peel oil were not perceived by panelists smelling of whole oil, or drinking orange juice. In conclusion, this study showed little difference between samples due to disease, either by GC-O or sensory evaluation. Only a few volatiles were perceived with greater intensity in the oils from healthy fruit in both Hamlin and Valencia, and three volatiles were only perceived in Valencia samples from HLB fruit. These differences were small and not important enough to be perceived in the oil by a sensory panel. However, in another study, cold pressed peel oil samples from Valencia fruit (26), each obtained from healthy, severely infected (HLBs) or mildly infected trees (HLBm), showed more, albeit similar differences. A total of 57 volatile compounds were identified by GC-MS in peel oil samples, including 9 monoterpenes, 16 sesquiterpenes, 12 alcohols, 13 aldehydes, 1 alkane, 2 ketones, 2 esters, and 2 terpene oxides. Of those, 14 compounds were found to be significantly different among healthy, HLBs and HLBm samples. Hexanal, (E,E)-2,4-decadienal, .-cadinene and .-copaene were significantly lower in HLBs samples than in the healthy samples, while sabinene, (E)-p-mentha-2,8-dien-1-ol, .-terpineneol, 3,7-dimethyl-6-octen-1-ol, (Z)-3,7-dimethyl-2,6-octadien-1-ol, carvone, cyclodecane, .-cubebene, (E)-.-farnesene, .-humulene and .-farnesene were significantly higher in HLBs samples. The contents of those volatiles in HLBm were in between. In conclusion for this study, HLB altered Valencia peel oil volatile profiles in that many terpenes were higher in HLB samples, probably due to disease stress upregulation of these compounds,while some aldehydes were surpressed, which may negatively impact the peel oil quality. However, sensory analysis was not done on these samples to confirm detectability.
Chemical and sensory analyses of fruit harvested from Huanglongbing (HLB) or greening-infected trees in winter (December Hamlin), 2012 (actually prior to start of project), winter (January, Hamlin) and 2013 spring (March and April, Valencia) were conducted. Trained sensory panels were completed and analyzed, while difference-from-control tests are still ongoing. Chemical analyses of sugars, acids, aroma volatiles, vitamin C, limonoids and flavonoids are completed, but the data not yet completely analyzed for aroma volatiles. The electronic tongue (etongue) analysis and molecular (qPCR) analysis of the titer of the pathogen (Liberibacter asiaticus) DNA in the juice (patent submitted) has been completed and analyzed. So far physical fruit measurements show that fruit from HLB-infected trees are smaller and more green than fruit from healthy trees, regardless of nutritional treatments of which 3 were investigated so far. Sugars, acids, ratio and total ascorbic acid were generally lower in HLB juice, although acids were sometimes higher in juice from symptomatic fruit, regardless of treatment, except for one nutritional treatment for Valencia in March, 2013. Bitter limonoids along with many other flavonoids were higher in HLB juice, regardless of nutritional treatment and especially in symptomatic fruit or fruit from severely infected trees. So far, the nutritional treatments have not shown a consistent effect, but there are sporadic positive effects on flavor chemicals. The nutritional treatments did not show an effect on reduction of Liberbacter titer in the juice as evidenced by qPCR analysis. The electronic tongue (etongue) and nose could discriminate between juices from healthy, asymptomatic-HLB and symptomatic-HLB fruit, with the etongue being much more effective. The etongue could also discriminate the different nutritional treatments within a harvest, but was confounded by seasonal changes across harvests. The etongue was more effective for Hamlin than Valencia, reflecting the more severe HLB-induced flavor effects for Hamlin. Trained panel showed differences in perception of orange and grapefruit, fruity, green and stale flavors and sweet, sour, bitter, metallic, tingling, astingent and umami (salty) tastes. The differences were minimized by nutritional treatments for Hamlin in December, 2012 and January, 2013, and sometimes nutritional treatments generally increased perception of sweetness. For Valencia, March and April, 2013 the nutritional treatments had no effect or enhanced sweetness but did not mitgate other off-flavors, respectively.
Fruit harvested from nutritionally treated or conventionally treated healthy or Huanglongbing (HLB) or greening-infected trees in January (Hamlin), 2014 and early and late April 2014 (Valencia with 3 new nutritional treatments added for a total of 9 that were replicated in the field). All analyses have been completed for the first year including trained and consumer sensory panels, chemical analyses of sugars, acids, aroma volatiles, vitamin C, limonoids and flavonoids and electronic tongue and the data analyzed. Juice samples were also analyzed by qPCR for Ct values to determin Liberbacter titer in the juice. So far physical fruit measurements show that fruit from HLB-infected trees are smaller and more green than fruit from healthy trees, regardless of nutritional treatments of which 3 were investigated so far for the earlier harvests. Sugars, acids and ratio were generally lower in HLB juice, and acids were sometimes higher in juice from symptomatic fruit, regardless of treatment, except for one nutritional treatment for Valencia in March, 2013. Bitter limonoids along with many other flavonoids were higher in HLB juice, regardless of nutritional treatment and especially in symptomatic fruit or fruit from severely infected trees, although this is more the case in early season harvests. So far, the nutritional treatments have not shown a consistent effect, but there are sporadic positive effects on flavor chemicals. The nutritional treatments did not show an effect on reduction of Liberbacter titer in the juice as evidenced by qPCR analysis so far for the first year. Several times, however one nutritional treatment has shown the ability to make the juice taste sweeter. The electronic tongue (etongue) and nose could discriminate between juices from healthy, asymptomatic-HLB and symptomatic-HLB fruit, with the etongue being much more effective. The etongue could also discriminate the different nutritional treatments within a harvest, but was confounded by seasonal changes across harvests. The etongue was more effective for Hamlin than Valencia, reflecting the more severe HLB-induced flavor effects for Hamlin in earlier samples, but the spring, 2013, Valencia samples did show separation indicating that the disease is becoming more severe for Valencia. Trained panel showed differences in perception of orange and grapefruit, fruity, green and stale flavors and sweet, sour, bitter, metallic, tingling, astingent and umami (salty) tastes. Consumer difference-from-control panels showed that the panelists detect differences that correlate to lower ratio and/or higher limonoids in HLB compared to healthy juice. The differences is greatest when there is both low ratio and high limonoids. Aroma volatile analysis through 2013 show that some tope notes, especially esters are lower in HLB juice generally, but more analysis is needed to determine nutritional treatment effects (unclear at this time). The general flavor differences were minimized by nutritional treatments for Hamlin in December, 2012 and January, 2013, and one nutritional treatment sometimes increased perception of sweetness. Samples were taken in recent harvests for analysis of peel oil and were processed but the samples have not all been run on the gas chromatograph nor the data analyzed. Mineral analysis on the first year juice showed elevated calcium in the HLB samples compared to healthy. So far, there are not consistent pattern for differences in ascorbic acid (vitamin C) due to HLB infection.
This portion of the project investigated the effect of HLB on Hamlin and Valencia orange peel oil quality by analyzing the volatile and olfactory profiles. In one study, peel oil samples were evaluated by gas chromatography-olfactometry (GC-O), where a person sniffs the volatiles as they come off a GC column, by GC-mass spectroscopy (MS) for peak identification, and by a sensory descriptive panel evaluating oil on filter paper. Analysis by GC-O revealed 57 odor-active peaks of which 33 were identified by GC-MS, and 22 confirmed by smelling chemical standards by GC-O. All identified compounds have been preciously reported in citrus and orange essential oil. There were 7 aliphatic aldehydes (hexanal, octanal, nonanal, decanal, undecanal, decadienal, dodecanal), 4 monoterpene aldehydes (citronellal, neral, geranial, perilla aldehyde), 3 aliphatic alcohols (hexanol, octanol, nonanol), 7 monoterpene alcohols (sabinene hydrate, linalool, 4-terpineol, citronellol, nerol, geraniol, cis-carveol), 8 monoterpene hydrocarbons (.-pinene, sabinene, myrcene, .-phellandrene, .-3-carene, limonene, .-phellandrene, terpinolene), 8 sesquiterpene hydrocarbons (.-copaene, .-cubebene, .-caryophyllene, .-copaene, .-humulene, germacrene D, valencene, .-cadinene), 1 monoterpene oxide (limonene oxide), 1 acid (methyl-octanoate) and 1 monoterpene ketone (carvone). Furthermore, 24 unknown compounds were detected by smell. Paired comparisons looked at Hamlin healthy/Hamlin HLB, and Valencia healthy/Valencia HLB. Multivariate statistics (PCA) found no differences in peak intensities between Hamlin healthy and HLB samples. Likewise, panelists could not distinguish between healthy and HLB samples for Hamlin oil. For Valencia oil, LRI 1185, .-cadinene and LRI 1645 were only perceived in HLB samples. Other differences were for LRI 955, .-phellandrene, and terpinolene, which had higher intensities in Valencia healthy. For GC-MS analyses, 10 compounds were significantly different between Valencia healthy and HLB, but contrary to Hamlin oils, they presented higher peak areas in Valencia HLB than in Valencia healthy, except for hexanal and cis-p-mentha-2,8-dien-1-ol. No significant difference was perceived between healthy and HLB Valencia samples by panelists during the difference test. Likewise, there were no significant differences between Valencia healthy and Valencia HLB juice made from the same oranges as for the peel oil extract. Therefore, the small differences detected by GC-O between healthy and HLB peel oil were not perceived by panelists smelling of whole oil, or drinking orange juice. In conclusion, this study showed little difference between samples due to disease, either by GC-O or sensory evaluation. Only a few volatiles were perceived with greater intensity in the oils from healthy fruit in both Hamlin and Valencia, and three volatiles were only perceived in Valencia samples from HLB fruit. These differences were small and not important enough to be perceived in the oil by a sensory panel. However, in another study, cold pressed peel oil samples from Valencia fruit (26), each obtained from healthy, severely infected (HLBs) or mildly infected trees (HLBm), showed more, albeit similar differences. A total of 57 volatile compounds were identified by GC-MS in peel oil samples, including 9 monoterpenes, 16 sesquiterpenes, 12 alcohols, 13 aldehydes, 1 alkane, 2 ketones, 2 esters, and 2 terpene oxides. Of those, 14 compounds were found to be significantly different among healthy, HLBs and HLBm samples. Hexanal, (E,E)-2,4-decadienal, .-cadinene and .-copaene were significantly lower in HLBs samples than in the healthy samples, while sabinene, (E)-p-mentha-2,8-dien-1-ol, .-terpineneol, 3,7-dimethyl-6-octen-1-ol, (Z)-3,7-dimethyl-2,6-octadien-1-ol, carvone, cyclodecane, .-cubebene, (E)-.-farnesene, .-humulene and .-farnesene were significantly higher in HLBs samples. The contents of those volatiles in HLBm were in between. In conclusion for this study, HLB altered Valencia peel oil volatile profiles in that many terpenes were higher in HLB samples, probably due to disease stress upregulation of these compounds,while some aldehydes were surpressed, which may negatively impact the peel oil quality. However, sensory analysis was not done on these samples to confirm detectability.
OVERVIEW The Budwood Certification Program continues to rebuild the Foundation and Increase tree collection. All trees in the Foundation and Increase screenhouses continue to grow and look good, while new trees continue to be propagated and added to the collection. All work has been completed to prepare all structures for TDA inspections under the new Citrus Nursery Stock Regulations that went into effect this fall. Inspections will begin in December, 2013. PROJECTS Increase Screen Structures: All Increase trees in the Screen Structures are doing well. All trees were pruned and hedged this fall. Pest inspections occur every week and all trees have been sprayed on a 2-4 week basis, applying insecticides, miticides and fungicides, both with soil drench thru the drip lines as well as foliar. Fertilizer has been applied on a regular basis both foliar and thru drip lines. Work has been completed on the structures to make sure they are ready for the official TDA inspections to begin in December. Foundation Screenhouses 1 & 2: The new Foundation trees planted in Screenhouses 1 and 2 are continuing to grow and look good. There are currently 86 trees (59 varieties) planted in-ground. Additional Foundation trees will be planted in early spring. Increase Screenhouse 3 & 4: Increase trees are continuing to be budded and transplanted in Screenhouse 3. Nearly 1,500 Increase trees have been transplanted and are active and another 1,000 trees were budded this October. Quotes for new tables for Screenhouse 4 have been submitted and a vendor is being selected by the University procurement office to purchase the tables. The table purchase should be completed in December. Foundation Screenhouse 5: There are currently 192 containerized trees (66 varieties) in Screenhouse 5. Additional trees will be added in early spring to bring it to capacity of 250. Buds from the Foundation trees in Screenhouse 1,2, and 5 as well as the Stephenville Greenhouse are used for new Increase trees going into Screenhouses 3 and 4. Stephenville Greenhouse: There are currently 83 trees (53 varieties) in the Stephenville greenhouse. More trees will be added this early spring to bring to capacity of 100 total trees. All trees look good. Fertilizer, pesticide and fungicides are applied regularly. PATHOGEN TESTING OF FOUNDATION & INCREASE TREES All Increase trees in the Screen Structures were root sampled and HLB testing on the root samples was completed. All trees tested negative for HLB. BUDWOOD SALES: Budwood sales to date for FY 2013-2014 are 28,798. Rio Red has accounted for 18,370 of total bud sales.
The microarray experiment to compare gene expression in salicylic acid deficient and normal plants following feeding by Liberibacter-infected or clean psyllids was completed and tissue samples including cauline leaves, rosette leaves and stems were collected for each plant. RNA was isolated from cauline leaves of 170 plants. Along with RNA isolations, phenotype data analysis was begun. We also initiated a new gene expression study to determine early responses of the NahG (salicylic acid deficient) and Col-0 (wild type) Arabidopsis lines. Plants were exposed to two treatments: infested with Candidatus Liberibacter psyllaurous (CLps) free psyllids and infested with CLps positive psyllids. In this study we plan to use qPCR to measure expression changes in known early disease response genes as well as candidate genes selected from results of the microarray experiment. This study was conducted in a similar manner as the microarray experiment except at 3wpi rosette leaves were collected from each plant for isolating RNA to be used for qPCR. Plants were grown in the same growth chamber and with identical growing conditions as in the previous microarray experiment. Phenotype observations and images were taken at each week. Another experiment was initiated to determine the effect on infection and symptom development of a mutation that makes plants insensitive to ethylene and thereby blocks the ethylene-dependent defense response pathway.
In our previous studies, we observed a novel symptomatic phenotype in Arabidopsis thaliana plants infected by Candidatus Liberibacter psyllaurous (CLps). This response appeared much more pronounced in plants of a transgenic line (NahG) that is salicylic acid deficient and compromised in its ability to initiate the salicylic acid defense response. To identify gene expression changes in Arabidopsis in response to Candidatus Liberibacter psyllaurous infection, a microarray experiment was initiated in December 2012 with the NahG and Col-0 (normal) lines. Plants were divided into three blocks and each block had plants from both the lines exposed to three treatments (non-infested, infested with CLps negative psyllids and infested with CLps positive psyllids). Starting at 3wpi (weeks post infestation), phenotype changes were tracked for each plant until 8wpi. For total of 216 plants (36 plants per treatment per line), the traits measured included the number of rosette leaves, length of rosette leaf, length of main stem, number of cauline leaves on main stem, flowers on main stem, siliques (fruits) on main stem, number of lateral stems, number of cauline leaves on lateral stems, number of flowers on lateral stems, and number of siliques on lateral stem. Observations such as discoloration on leaves and leaf curling on plants were also noted each week post infestation. Images of all plants were taken each week for all groups. At the end of the experiment (8wpi), tissue samples were harvested from each plant for RNA and DNA isolation for microarray and pathogen detection respectively. For the microarray analysis, plants exposed to CLps-infected psyllids will be divided into symptomatic and asymptomatic groups to give 4 treatments in all. This study will enable us to not only understand Arabidopsis responses to CLps but can also provide a well studied model plant species for HLB-related research.
Preparation of samples for a microarray experiment to compare gene expression in salicylic acid deficient and normal Arabidopsis plants following feeding by Liberibacter-infected or clean psyllids continued. We determined the quality of RNA isolated from 170 plants using a Bio-analyzer. Good quality RNA samples were pooled within treatment x block combinations to reduce biological variability. Along with processing RNA samples, we continued analysis of phenotype data collected during this time. We found significant differences among treatments in a number of traits including the number of rosette leaves, number of plants with discoloration, number of plants showing young leaves curling and increased number of cauline leaves among the treatments. The number of rosette leaves was significantly lower in infested plants (Infested with CLps-negative psyllids and CLps positive psyllids) in comparison to non-infested plants. There was significantly high number of plants infested with CLps positive psyllids showing discoloration and a phenotype of new leaves curling in comparison to the other two treatments for normal and salicylic acid deficient lines. Statistical analysis also showed that the number of cauline leaves was significantly higher in plants infested with CLps positive psyllids. Thus the preliminary analysis of this data indicates that feeding by psyllids (whether clean or CLps-infected) reduces plant growth slightly. Feeding by infected psyllids increased the frequency of leaf curling, purple coloration, and production of cauline leaves. In previous experiments not all plants inoculated with infected psyllids became qPCR positive. We have not yet conducted the qPCR tests (over 1000 samples) to distinguish infected from non-infected plants but it seems likely that separation of qPCR-positive and qPCR-negative plants within the CLps-inoculated group will improve our ability to interpret the results of this experiment. Data collection was completed for an experiment to identify early gene expression of normal and salicylic acid deficient lines.
“The main aim of this project is to express molecules in plant that interfere the growth and ACP- transmission of CLas ” Brief description: Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication systems. But the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs We detect AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. Main findings: 1-Using different bacterial biosensor, we partly identify these AHLs (number of Carbon). CLas biofilm formation on the surface of insect Gut confirms the presence of cell-to-cell communication in insect while the planktonic state of CLas in plant indicate the absence of this communication. 2- In plant, we found molecules that bind to LuxR but inactive its function (plant defense). We try now to characterize these molecule and study their effect on biofilm formation inside insect. We use purified molecule to feed infected insect through artificial diet system. 3-We produced citrus plants that express LuxR protein in the phloem sap in order to test I- If the acquired LuxR proteins in insect interfere with the biofilm formation in insect (cure the insect from CLas) II- if the expression of LuxR in plant induce biofilm formation (localize the infection in plant) We found that feeding infected ACP with CLas on the LUXR expressing plants reduce the bacterial populations in insect and reduced the infection rate significantly. This result strongly indicates that we can target this system to interfere with the insect transmission and the spread of Disease. In last few months we focused on identifying the AHL- like molecules in plant Phloem sap. For that were analyzed the phloem sap chemical composition from suspectable and resistant varieties of citrus. “Finding for this report: We have identified molecules that are structurally non-related to AHL but reported in other system for their capacity to bind to bacterial LuxR such as GABA and Riboflavin like compound. We are testing these comound for their ability to bind to CLas-LuxR and induce the GFP fluorescence using our E. coli-LuxR biosensor. The main aim of this project is to express molecules in plant that interfere the growth of CLas in insect by feeding.
Brief description: ‘The efficiency of transmission process of Candidatus Liberibacter asciaticus (CLas) depends on the success of specific interactions between CLas and the insect vector Asian citrus psyllids (ACP). CLas is circulative propagative within ACP. The bacteria need to pass the intestinal barrier to reach the hemolymph where they multiply then they must invade the salivary glandes in order to be inoculated in a new plant host while insect feeding. Passing these biological barriers needs specific interactions between CLas cells and the epithelial cells in the guts and the salivary glands cells.’ Methodology: The main technique we use in our study is the protein overlay assay. In this method, non CLas-carrying insect total proteins were separated by 2D-SDS-PAGE. After 2D-SDS-PAGE, the proteins were blotted onto PVDF membrane. the membrane was overlaid with extract from infect citrus then we detect the complex using antibodies against CLas membrane proteins. this method called (Far-western) Major findings We were successful in identifying the receptors in the Asian citrus psyllids (ACP). The receptors are the proteins that CLas cells recognize and bind to before passing the gut to the hemolymph. Same thing, when cells invade the salivary glands to reach the food canal. Comparisons between the stained electrophoretic profiles of ACP proteins in the gel and results of far-Western blot experiments on the membrane allow to cut the protein spots from 2-DE gels for LC-MS/MS analysis. Some ACP proteins (receptors) were identified. The function of these proteins was analyzed with bioinformatics. These genes were cloned, proteins were expressed, antibody against these proteins were made. “a publication described this work is in preparation” Recently, we started to identify the membrane proteins from CLas that interact and bind to the identified receptors. Since we have identified the receptors in ACP, we are performing negative FAR-Western to identify the ligands in CLas. For this reason, we predicted the antigenic domains in the identified ACP-receptors to produce the antibodies. We already obtained antibodies against some of them. Our aim for the next few months is to identify the proteins of Clas (ligands) that adhere to ACP cells We started purified the membrane proteins from infected phloem sap (CLas membrane proteins) using TritonX114, X100. investigated proteins in PAGE show very nice membrane protein profile. We will use these purified membrane proteins for our negative FAR-Western The yeast double hybrid system will be used to validate specific interaction for each couple (receptor-ligand) Overall, our research work is carried out according to milestone of the project.
The transformation construct for expressing the FLT-antiNodT fusion protein in citrus has been completed. We encountered major difficulties cloning the FLT-antiNodT expression cassette into the pTLAB21 citrus transformation vector. The FLT-antiNodT cassette DNA was unstable in E. coli when cloned into pTLAB21, which stymied our cloning efforts for several months. The instability of the FLT-antiNodT cassette in pTLAB21 was surprising, since the FLT-antiNodT cassette was stable in E. coli when cloned into non-transformation vectors such as pBluescript. For reasons unknown, the FLT-antiNodT cassette was specifically unstable in pTLAB21. However, we serendipitously discovered that the inclusion of an additional segment of DNA next to the FLT-antiNodT cassette in pTLAB21 actually stabilized the FLT-antiNodT cassette in pTLAB21. This piece of DNA was derived from the original FLT-antiNodT cassette cloning vector, pBluescript. This additional piece of DNA will not cause a problem for transformation of citrus or expression of the FLT-antiNodT antibody in transgenic citrus. We have performed DNA sequencing of the pTLAB21-FLT-antiNodT transformation vector and have verified its identity, stability in E. coli, and sequence integrity. The pTLAB21-FLT-antiNodT transformation vector was sent to the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The pTLAB21-FLT-antiNodT transformation vector was introduced into Agrobacterium tumefaciens strain EHA101. PCR was used to verify that the pTLAB21-FLT-antiNodT transformation vector was intact in EH101. Transformation of ‘Duncan’ grapefruit was initiated in late October, 2013.
1. The binary vector for an inducible cre-lox based marker free selection has been constructed containing a heat inducible excision system containing the cre gene driven by a Soybean heat shock gene promoter. Tobacco has been transformed with the construct and we are observing a 35-50% excision of the selectable marker gene in the regenerated plants. Citrus epicotyl explants will be transformed with the same construct as seeds become available this fall. Transgenic plants containing a putative Citrus sinensis (sweet orange) small heat-shock protein gene promoter are also being generated. 2. Hamlin and W. Murcott cells have been transformed with a binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cell. Our data indicates that the transformation vector is functional and able to incorporate both T-DNAs into the plant genome. Putatively transgenic somatic embryos have begun to germinate and will be tested by once the plants are large enough. Experiments are being conducted to confirm if negative selection pressure can differentiate between cells that contain the marker free T-DNA from the T-DNA containing the selectable positive/negative fusion marker cassette and if it can be removed from the citrus genome. 3. The citrus FT gene has been incorporated into Carrizo citrange. Numerous transgenic plants containing the Citrus FT stacked with the citrus AP1 have also been produced for testing. Plants are growing in the laboratory and will be tested for the presence of the gene when they reach suitable size. 4. PCR analysis of transgenic plants containing the NPR1 gene stacked with the CEME transgene have identified 7 lines that contain both transgenes. These transgenic lines are being grown in the greenhouse for cloning and testing. Agrobacterium mediated transformation to produce more lines will commence in the following quarter. 5. Targeting AMP expression in the phloem: we have produced additional transgenic plants using Agrobacterium-mediated transformation (Duncan, Carrizo, Pineapple, Hamlin, and Valencia) with theLIMA gene controlled by the phloem limited AtSUC2 promoter. Some transgenic lines from these cultivars have been propagated for further characterization and molecular analysis is underway. 6. Transgene expression and correlation to disease resistance response: ‘ Western analyses for LIMA and GAN transgenes is nearly completed – transgenic plants from independent transformation events show quite variable transgene expression as expected.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. Since we are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, we changing our focus of building new constructs to controlling psyllids until we have more conclusion from the peptides under screen. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences.
Using the heating tunnel prototype built last year, which incorporates two infrared radiant heaters and four fans, 36 trees were heat treated at three different temperatures. Within each treatment temperature, three different duration times were also tested. Each of these nine combinations were repeated four times and randomly assigned to HLB infected trees. During these experiments a third heater was added to the prototype for use during the higher temperature tests. It was necessary for the temperature to increase more rapidly during these tests. The third heater also helped maintain the higher temperatures more efficiently. Two more trees were randomly selected for testing an extremely high temperature over a very short period of time. For this experiment, the third heater radiant heater was added. Reflective panels were also placed on the ground below the tree to aid in maintaining such high temperatures. This test was added to evaluate the maximum heat treatment that can be applied before permanent damage to the tree occurs. A heat transfer test was conducted to quantify and model the amount of heat the tree will absorb during each test and to measure the heat distribution throughout the tree canopy and within the tree. Small wholes were drilled at varying parts of the tree and thermocouples were placed inside. Surface temperatures and air temperatures were also collected at these sites. This test was done using the same heating tunnel prototype with some minor adjustments. The third heater was added and reflective panels were placed on the ground to limit the heat absorbed by the soil. This test will later be used as a tool to design a model for the transfer of heat into the tree during each test. To further quantify the results for tree health, physiological measurements were added to the study. These measurements include porometer readings, pressure readings, and leaf anatomy samples. For each time-temperature combination, two trees were randomly selected for these tests. Samples were taken before treatment, one week after treatment, one month after treatment, and will continue to be tested throughout the remainder of the experiment. Trees tested at lower temperatures show less signs of physical change having almost no defoliation or fruit drop. Trees tested at the higher temperatures however, were almost completely defoliated and have started to produce new growth. The prediction is that these trees will show the greatest response to the treatment. Even the trees tested with an extremely high temperature are showing signs of new growth which shows that the trees may be able to withstand even higher temperature treatments.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
