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. Preharvest fruit drop was monitored in a Hamlin (50% on Swingle, 50% on Carrizo) block located in Lake Alfred, FL from August to December 2013 with the final fruit drop count performed one week before harvest. At harvest the total remaining fruit on the tree were estimated to calculate a true percentage of the fruit dropped prior to harvest. The mean preharvest fruit drop for the block was approximately 42%. Data analysis is currently underway and suggest that preharvest drop was approximately 10% higher in trees with Carrizo rootstock compared to Swingle. We do not anticipate any significant differences between the plant growth regulator treatments with respect to preharvest fruit drop although additional statistical analysis is required due to the high variability in tree health within the trial. The harvested fruit was analyzed for size and quality. Fruit were smaller than average for all treatments and there were no significant differences between treatments. Phloem functionality data is currently being evaluated along with other measurements that will be available in a future report.
A number of genetic constructs have been received from various scientists, with and without supporting documentation (i.e. plasmid maps, sequence, publications). The genetic constructs for which we have supporting documentation have been put in the queue. The constructs without supporting documentation have been appropriately stored until information is received. As a new step in quality control, we are sequencing some plasmids, particularly those lacking the pVS1 replicon for stability in Agrobacterium. A genetic construct obtained from Dr. Mou has been transformed into mature scions of Hamlin, Valencia, Ray Ruby and mature rootstocks of Carrizo and Swingle. As advised, we have discontinued work with Pineapple sweet orange. Shoots will be pre-screened once they are bigger. Transgenic shoots of Valencia or Hamlin be micro-propagated and budded in different combinations with transgenic or wild-type Carrizo or Swingle immature rootstock. These trees will be submitted for disease screening to determine which have superior disease tolerance and whether transgenic rootstocks confer graft transmissable tolerance. Different cytokinins will be tested in scion and rootstock micro-propagation. Progress in being made towards increasing the productivity of the lab and the growth room. In the lab, mature scion and rootstock stem explants are being cut to 0.6 cm rather than the standard 1.0 cm for tissue culture. These smaller explants survive the Agrobacterium transformation protocol well and regenerate plantlets. This approach increases the explants available for each weekly transformation from ~600 to 1000. In the growth room, rootstocks are being budded with mature scion at an earlier age and the results look promising. Using this approach, we only need to transplant the rootstock on which the mature scion bud has opened. In an additional effort to increase our productivity, small experiments are being conducted to determine whether we can regenerate shoots from calli derived from mature leaf tissue after Agrobacterium treatment. Plantlets have been regenerated from the calli in one variety and are being elongated. These plants should still flower and fruit early because they have undergone the phase transition from immature to mature. We have also been able to root some varieties. NPTII immunostrip tests were conducted in sweet orange and grapefruit trees transformed with marker genes. Out of 47 transgenics tested, 34 tested positive for expression of the NPTII gene. Genomic DNA extraction and Southern blots to show transgene integration are underway.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
Two different cytoplasmic viruses (CiLV-C and CiLV-C2) are now know to cause citrus leprosis. In addition a virus found in the nucleus, nuclear citrus leprosis virus (CiLV-N), also can cause citrus leprosis disease in North and South America. All types of the leprosis viruses are transmitted by Brevipalpus mites. We have 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. Mite transmission experiments with the citrus leprosis affected citrus leaves from Mexico (CiLV-N) & Colombia (CiLV-C2) were done and two months after completion of the transmission experiments none of the citrus seedlings showed symptoms of leprosis. Leaf tissue from the experiments were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using CiLV type-specific primers and were negative. Using newly developed PCR primers we were able to prove the viruliferous status of Brevipalpus mites taken from infected lesions, killed in ethanol and shipped from Mexico (CiLV-N) and Colombia (CiLV-C2). We are continuing the use of these PCR primers to determine if Florida endemic healthy Brevivalpus Florida mites acquire the various citrus leprosis viruses. Additional collection of 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 Guillermo Leon, continued work on the transmission and interactions of of CiLV-C2 and the vector, Brevipalpus phoenicis (Geijskes). The acquisition of the virus from citrus, then feeding on non-citrus host plants (some are hosts of the virus) and then feeding on citrus plants for virus transmission was evaluated. The appearance of the leprosis symptoms on Valencia orange plants was14-37 days after feeding, when the mites had previously fed on Swinglea glutinosa, Dieffenbachia sp. and Codiaeum variegatum for 2 to 20 days. When the mites were allowed to feed on Sida acuta, leprosis symptoms occurred in oranges between 18 and 46 days and when the mites fed on Hibiscus rosa-cinensis and Stachytarpheta cayennensis the symptoms appeared between 26-51 days after transmission. In conclusion it appeared that feeding on non-citrus hosts had no effect on virus transmission.
“This project aims is to express molecules in plant that Disrupt the growth and ACP- transmission of CLas ” project narrative: 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 since the start of project: 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. 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 compound for their ability to bind to CLas-LuxR and induce the GFP fluorescence using our E. coli-LuxR biosensor. IN THIS REPORT: we studied the phloem sap composition of 14 different citrus varieties by GC-MS after derivitization with 2 different reagent to increase the limit of detection. we are analyzing these data right now to look for similarity in the composition in tolerant varieties and in susceptable varieties. this study will give us idea about what the citus phloem sap provide CLas with that may play as a signaling factor. We try to confirm our previous finding (GABA and Riboflavin like compound) and to link the presence of these compound with the susceptibility.
Project narrative: We aim to understand the specific interactions between Candidatus Liberibacter asciaticus (CLas) and the insect vector Asian citrus psyllids (ACP) to block the transmission. ‘The transmission process of CLas depends on the success of specific interactions between CLas and the insect vector ACP. The bacterium passes through 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.’ major finding since the start of project: 1- Identification of the receptors in the Asian citrus psyllids (ACP). 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” 2- Recently, we were able to establish a new method we called it Reverse Far-Western to identify the membrane protein in CLas that bind to the receptors. “Since we have identified the receptors in ACP, 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 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 reverse 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. Currently, 1- We are purifying Ferritin from ACP haemolyph to check if it bind to the CLas membrane protein. a potential role for ferritin as a CLas carrier is found in our work. 2- We start to establish the complexome to study the interaction of both partners (receptor-ligands) in native conditions. 3- We are targeting ACP receptor by RNAi to test if we can interfer with the interaction with CLas and subsequently the transmission.
There was no funding received until the end this quarter, so little progress could be made.
This project has been difficult from the beginning and when we were coming up on the third year, all of my Co-PIs decided they did not to continue on this project for various reasons. I did want to continue because my part of the project was going well. It took some time to sort this out but by May 9, 2012 I had e-mailed Dr. Browning for guidance. He indicated that I could submit by myself. I did this on June 19. I know it was received because Ms. Nowicki had me change something in the budget. And the executed contract was sent. Then I heard nothing. On September 28, Dr. Turpen called me about why I had not submitted paperwork. I told him I had and resent the third year paperwork to him. Again I heard nothing until an NOA was sent in the middle of November for $39,000. When someone in DSR asked Ms. Nowicki about a no-cost extension, she said there would not be one, I had until December 31 to spend the money because I had been so late in getting in the paperwork. I was not extremely late in getting in the paperwork! And of course I could not spend all of the funding in six weeks (during the holiday season) without doing things I am not comfortable doing because I have no desire to fail an audit or get in trouble with either CRDF or UF. A 6 month no-cost extension was finally executed. This allowed us to continue preliminary work on cell penetrating peptides, which led to the funding of Project 752. How to write the reports was problematic because of the gaps in funding and although I asked for guidance, I did not receive any.
There was no funding received during this quarter, so progress could not be made.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated.
Oral uptake of dsRNA targeting specific Asian citrus psyllid genes can induce psyllid mortality and reduce Liberibacter titer in infected psyllids. Significant progress has been made with research on the use of RNAi as a means of Asian citrus psyllid control. We have made use of a Citrus tristeza virus (CTV) dsRNA expression system which when inoculated into C.Marcrophylla results in leaf phloem containing dsRNAs which target essential ACP genes. When ACP feed on leaves from these “paratrangenic” (CTV transfected) citrus plants, mortality was double that observed when the ACP fed on artificial diets containing this synthetic dsRNA, greater than 80% mortality. Currently, new versions of paratransgenic citrus are being produced to determine the best sequence to use targeting this essential gene. The use of the Ion Torrent (by Life Technologies) next generation sequencing has provided rich insight into the transcription profile of “paratransgenic” fed “sick” ACP using comparative RNA Seq analysis. The data support the specific down-regulation of the dsRNA target gene as the cause of mortality as seen by the significant perturbation of genes in the molecular pathway in which this gene functions. RNA Seq data continues to be collected from ACP fed on a variety of gene specific dsRNA containing diets. Experimentation has also begun using artificial diets containing dsRNAs synthesized by a novel “mass-production” technique that would make it practical for the use of RNAi technology as a field application. The use of topical application in conjunction with “paratransgenic” plant varieties presents a strategy for effective delivery and a multiple gene targeting employment of the RNAi pest control technology.
We hypothesized that groves with high bicarbonate stress are suffering from HLB because they support lower fibrous root density compared to groves with lower bicarbonates (less than 100 ppm) in irrigation water and/or soil pH (less than 6.5). To confirm this relationship, we surveyed 37 grove locations in Highlands and Desoto counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is significantly related to well water pH greater than 6.5 and to soil pH greater than 6.2. Yield records from these blocks reveal that groves under high bicarbonate stress production have declined 20 percent over the last three seasons (2009-2012) in contrast to Ridge groves with low bicarbonate stress which have increased 6 percent in production even though HLB incidence has accelerated. The yield losses are correlated with less fibrous root density, which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicates that bicarbonate impairs the root’s ability to take up important nutritional cations including Ca, Mg and K, as well as micronutrients, especially Mn and Fe. In Florida, Bryan Belcher of Davis Citrus Management has acidified irrigation water with sulfuric or N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in the same way as fertigation. N-furic has the advantages of being safer to handle and providing some additional N due to the urea component, but the disadvantage is higher cost of treatment compared to sulfuric acid. Since irrigation is not necessary when it rains, acidification treatment only occurs during the dry season when the bicarbonates are loading into the wetted area under the tree. When the rain begins, these bicarbonates are flushed from the rhizosphere. Our labs and grower cooperators are also evaluating acidification of soil by amendment with elemental sulfur applied in prilled or finely ground form. Sulfur (S) releases acid when it interacts with Thiobacillus bacteria in soil to form acid (H+) ions. This process of acidification with S is slower than treatment of the water but provides for longer lasting reduction in soil pH. Sulfur can be applied in prilled form with a fertilizer spreader or with a herbicide boom as a slurry. Sulfur can also be added to dry and fertigation formulations to lower the pH by as much as one unit after repeated ground applications.
We hypothesized that groves with high bicarbonate stress are suffering from HLB because they support lower fibrous root density compared to groves with lower bicarbonates (less than 100 ppm) in irrigation water and/or soil pH (less than 6.5). To confirm this relationship, we surveyed 37 grove locations in Highlands and Desoto counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is significantly related to well water pH greater than 6.5 and to soil pH greater than 6.2. Yield records from these blocks reveal that groves under high bicarbonate stress production have declined 20 percent over the last three seasons (2009-2012) in contrast to Ridge groves with low bicarbonate stress which have increased 6 percent in production even though HLB incidence has accelerated. The yield losses are correlated with less fibrous root density, which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicates that bicarbonate impairs the root’s ability to take up important nutritional cations including Ca, Mg and K, as well as micronutrients, especially Mn and Fe. In Florida, Bryan Belcher of Davis Citrus Management has acidified irrigation water with sulfuric or N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in the same way as fertigation. N-furic has the advantages of being safer to handle and providing some additional N due to the urea component, but the disadvantage is higher cost of treatment compared to sulfuric acid. Since irrigation is not necessary when it rains, acidification treatment only occurs during the dry season when the bicarbonates are loading into the wetted area under the tree. When the rain begins, these bicarbonates are flushed from the rhizosphere. Our labs and grower cooperators are also evaluating acidification of soil by amendment with elemental sulfur applied in prilled or finely ground form. Sulfur (S) releases acid when it interacts with Thiobacillus bacteria in soil to form acid (H+) ions. This process of acidification with S is slower than treatment of the water but provides for longer lasting reduction in soil pH. Sulfur can be applied in prilled form with a fertilizer spreader or with a herbicide boom as a slurry. Sulfur can also be added to dry and fertigation formulations to lower the pH by as much as one unit after repeated ground applications.
Stress intolerance of HLB trees is a direct consequence of a more than 30 percent loss of fibrous root density compared to non-diseased trees. This root loss may be compounded by interactions with root pathogens and pests such as Phytophthora spp. Progagules of Phytophthora spp. are elevated in the rhizosphere soil of HLB trees. When Las interacts with Phytophthora, fibrous root loss can be greater than that caused by HLB alone, depending on the grove location and time of year. Although not directly studied, HLB associated root loss has been attributed to carbohydrate starvation. Initially, canopy symptoms are not apparent in trees with significant root loss, so starch concentration in roots was determined. Surprisingly, early root loss occurred without a drop in starch. This disappearance of roots could be due to the lack of new growth to replace old roots during the normal cycle of death and regeneration of fibrous roots, or may be associated with premature root death unrelated to phloem plugging, or a combination of the two processes. Also, it is highly likely that Las damage alters the concentration of soluble sugars in roots by increasing leakage from roots that attracts and accelerates infection by root pathogens such as P. nicotianae.
Seasonal root sampling continues in two field sites with a third site identified. Sampling has already revealed seasonal variation in root infections and apparent shifts in the root flush cycle caused by Liberibacter. We have swapped out root cages twice and collected data on root flushes in response to HLB during a normal root flush and nonflushing time. Data analysis on initial root flush data in comparison to overall root density is underway and data collection will continue through the year. Some difficulty is being encountered with finding sufficient presumed healthy trees in the field, so more emphasis will be placed on greenhouse experiments as the project continues. We will continue to look for new sites with moderate infection rates and trees old enough for routine root sampling. Sampling at a rootstock trial site is underway with a full year of data on the effects of HLB on these new experimental rootstocks. This has already begun to demonstrate how these new rootstock lines respond to Liberibacter infection. We are in the final preparation stage of testing the most promising rootstock lines in more controlled greenhouse studies and are awaiting improved weather conditions for high efficiency inoculation Liberibacter.
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