In this proposal our objective is to find citrus versions for the two proteins that make up the functional components of a chimeric antimicrobial protein (CAP) previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have successfully identified a suitable replacement for the first component, the human neutrophil elastase (HNE) that also serves as the surface binding component of CAP. Since HNE is a serine protease with elastase activity whose 3D structure has been determined we used the PDB database to find a suitable plant protein with the same 3D structure. Using the active site geometry of HNE is a consistent structural feature we focused on a set of 288 non-redundant plant derived proteins extracted from the PDB database to narrow our search criteria. The key feature of our search involved using CLASP to search for a match using the electrostatic properties and structural geometry of the three amino acids that make up the active site of HNE. We obtained a close match with the tomato PR14a protein. Using the tomato amino acid sequences we then searched for a similar citrus protein by searching through citrus genome information in Phytosome (http://www.phytozome.net). This was successful and we have identified a single protein that has the identical amino acid sequence in both Citrus sinensis (Cs) and Citrus clementina (Cc) genomes. We have focused the 165 amino acid P14a protein from Cs which we refer to as CsP14a. We have analyzed this sequence and have determined that it is a secreted protein and contains what appears to be a 25 amino acid signal sequence. We have utilized the 137 aa mature protein and successfully constructed two synthetic genes that encode this protein, one that just contains the coding region of CsP14a and the other is a chimeric version that contains the CsP14a coding region linked to the CecropinB (CecB) protein. We have included a signal peptide (22aa) that we have used before and know works really well at secreting proteins to the plant apoplast and xylem. This signal peptide has been added at the N-terminal of this protein and we have added a Flag Tag also at the N-terminal so that the protein can be easily detected and purified. The Flag tag will remain a part of the secreted protein after cleavage of the signal peptide. We are constructing two CaMV35S expression cassettes to express both these synthetic genes. We are using CLASP to identify a citrus replacement component for CecB. Since CB has no enzymatic activity, we could not use a well-constrained motif like an active site. We chose instead the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. Our analysis has proved fruitful and we have identified a good plant candidate that has the same shape and that is highly conserved in plants.
Our accomplishments are: 1) Various young and mature citrus plants and also citrus seeds that are used for this project were purchased, planted and maintained in a greenhouse; 2) Sterile culture of citrus plant materials were established; 3) Construction of the proposed genes that should enhance shoot regeneration and embryogenesis has been started and is well underway.
A gene obtained from Dr. Mou that confers tolerance to canker has been transformed into mature Hamlin, Valencia, Pineapple, Ray Ruby, Carrizo, and Swingle, and shoots are regenerating. Unfortunately a number of PCR positive Pineapple shoots that were micro-grafted onto Carrizo rootstock and maintained in liquid media were lost due to an error in media preparation. This error has been corrected. Six shoots of micro-grafted Pineapple survived from this batch and an additional 33 Pineapple shoots have been micro-grafted onto Carrizo rootstock. Shoots regenerated from Carrizo and Swingle explants are being elongated in tissue culture prior to rooting. GUS assays for the reporter gene and additional molecular analyses will be conducted once the shoots are larger. Two constructs obtained from Dr. Wang were also used in transformation experiments of mature Valencia. Shoots were micro-grafted onto Carrizo rootstock and are still growing in liquid media prior to secondary grafting or have already been transferred to the soil. Molecular analyses will continue once the plants are larger. Molecular analyses of Hamlin, Valencia, Pineapple, and grapefruit scion transformed with marker genes are underway. Thus far, nine out of ten GUS or GFP positive plants have tested positive for the expression of the nptII transgene using the nptII immunostrip assay. The remaining ~40 transgenics will be tested using nptII immunostrips, nptII ELISAs. and Southern blotting. The first flower buds have formed on a Valencia transgenic event originally generated on 5/30/12, so it has been ~19 months for flowering to occur which agrees with Dr. Pena’s protocol. One of the limitations of the mature scion transformation protocol is the relatively slow process of bud break of the scion following grafting and the slow growth of the scion for explants. The double budding procedure and daily hormone applications to induce early bud break have significantly increased productively of the growth room. Buds now break one week after the grafting tape has been removed. We have observed significant differences in bud break of the scion on different rootstocks following hormone application. The photoperiod has been extended to 19 hours of light and 5 hours of dark to further increase the rate of vegetative growth and productivity of the growth room. A number of scientists were contacted to provide additional constructs and three scientists indicated they will provide constructs in the near future.
Cytoplasmic (CiLV-C and CiLV-C2) and nuclear (CiLV-N) citrus leprosis virus cause citrus leprosis disease in North and South America. All types of the leprosis viruses are transmitted by Brevipalpus mites. We continued mite transmission experiments at the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD with endemic healthy Brevivalpus yothersii (syn. phoenicis) mites from Florida. We again did mite transmission experiments with the citrus leprosis affected samples from Mexico (CiLV-N) & Colombia (CiLV-C2). As reported previously six weeks after completion of the transmission experiments none of the citrus seedlings showed leprosis symptoms. For confirmation of the negative test results leaf tissue from the experiments were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using CiLV type-specific primers. Recently, nuclear CiLV was reported from Mexico but no prior sequence information was available. We successfully determined the entire genome sequence of nuclear CiLV and and published a manuscript on this sequence in the journal Genome Announcement (‘Genome assembly of citrus leprosis virus nuclear type reveals a close association with orchid fleck virus’. Our collaborator in Mexico has shipped us further shipment of infected nuclear leprosis samples to continue transmission experiments. Results are pending on these transmissions. Using newly developed PCR primers we are hoping to determine the viruliferous status of Brevipalpus mites in acquisition of the nuclear citrus leprosis virus. Based on these results we will determine if Florida endemic healthy Brevivalpus Florida mites are able to acquire the various citrus leprosis viruses. In addition mites (preserved in alcohol) from Mexico have been sent to USDA cooperators to continue to compare their taxonomic status with those that do transmit in Colombia and elsewhere. In Colombia our cooperator, Guillermo Leon, continued work on the transmission, viability and interactions of the of CiLV-C2 and the vector, Brevipalpus phoenicis (Geijskes). The acquisition of the virus from citrus, then feeding on non-citrus host plants and returning to citrus plants for virus transmission was evaluated. Results were that B. phoenicis mite was able to transmit the virus to Valencia orange plants (Citrus sinensis L.) at a transmission rate of about 80%, after having fed for periods of two to twenty days in any of the six alternate mite host plants. The alternative host plants included Dieffenbachia sp., Hibiscus rosacinensis, Codiaeum variegatum, Swinglea glutinosa, Sida acuta or Stachytarpheta cayennensis. The appearance of the first leprosis symptoms in the Valencia orange leaves, when the mite previously fed on S. glutinosa, Dieffenbachia sp. and C. variegatum appeared the earliest followed by S. acuta and then followed by H. rosacinensis. All of these results have been submitted for publication.
The goal of this experiment is twofold, first to determine the effects of plant growth regulators on addressing vascular degeneration and fruit drop, and second to determine the effects of HLB and ACPS citriculture on drought tolerance. A field experiment was installed in April 2013 to test the efficacy of the synthetic auxin 2,4-D and a micro-emulsion ‘based surfactant to reduce HLB symptom severity in a mature ‘Hamlin’ orange block. The HLB incidence in the block is currently more than 50% and consequently the fruit yield losses due to pre-harvest fruit drop from symptomatic trees were devastating in the 2012/13 season. The experimental design is a 4×4 Latin Square with four replications and four factorial foliar spray treatments consisting of 2,4-D, Eco-Agra’ surfactant, 2,4-D + Eco-Agra’, and untreated control. Each whole plot is split into two sub-plots containing Swingle and Carrizo rootstocks. A basal foliar nutrient spray treatment applied to the whole experiment consists of a comprehensive, balanced fertilization program of micronutrients, macronutrients and potassium phosphite products timed to coincide with the major leaf flushes. The basal ground-applied fertilizer program consists of a dry granular bulk-blended N-P-K +Ca +Mg + Fe + Mn +Zn +B +S product applied four times in the growing season. The automated micro-sprinkler irrigation system is used to apply water to the trees according to seasonal evapotranspiration demand as needed, up to twice per day. We have been monitoring preharvest fruit drop in this 4-acre Hamlin block in Lake Alfred since 8/19/2013 every two weeks by raking out the dropped fruit from 4 trees per block, 2 trees on Swingle and 2 trees on Carrizo to determine the efficacy of the 2,4-D/EcoAgra treatments. The control and 2,4-D+EcoAgra treatments appear to be having the most significant fruit drop, with an average of 200-215 pieces of fruit dropped per tree. The EcoAgra or 2,4-D treatments on their own appear to be reducing drop, but only by about 35%. These data are preliminary and should not be used as a recommendation for growers at this point. Within the next two weeks the fruit from the experimental trees will be picked and we can then determine the percentage of fruit dropped prior to harvest for individual trees. This will be extremely important due to the high variability of symptoms in this block. For example, several trees entered the experiment with moderate to severe HLB symptoms and over the past 4 months have completely defoliated and dropped all of their fruit. Meanwhile, other trees remain vigorous with mild HLB symptoms and a good crop load.
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.
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