The modifications to the citrus economic investment model and the citrus reset model to enable comparison of the enhanced foliar nutrient program to the traditional HLB management program were completed. An economic analysis using these models was done that estimates the annual HLB infection rate that switching from the traditional HLB management program to an enhanced foliar nutrient program maximizes profits. The results of this analysis will be presented at the 2nd International Research Conference on HLB in Orlando, Florida in January, 2011. Prior to the discovery of HLB, or citrus greening, in Florida in 2005, resetting diseased/unproductive trees was usually the most economically viable strategy to maximizing the economic life of citrus groves. In some situations, replanting the grove was required but the replanting decision was obvious since the grove had probably been destroyed from a freeze, hurricane or been removed as part of the canker eradication program. However, the introduction of endemic greening into Florida citrus groves has made the grove replacement decision critical to maximizing the profitability and economic life of citrus groves. It is assumed that the grower’s objective is to maximize the net revenues generated by the operation of a citrus grove and its replacements over their lives. For grove replanting decisions, this objective is best accomplished through marginal analysis, where the net revenues from operating the grove for another season are compared with the opportunity to earn higher future net revenues that would be initiated by replanting the grove during that season. The model has been developed and is now in preliminary form. It is designed to estimate the net revenues at which citrus groves infected with HLB should be replanted and what type of technologically improved grove to replant. The data used in this analysis were from a commercial grove planted to a density of 270 trees per acre in the late 1980’s. Expected fruit prices, yields, tree attrition rates, and operating costs for both the existing grove and the replacement grove are all key variables that affect optimum replacement times. Also, capital required for replanting and any other variables that will affect the performance of the replacement grove must be included in the analysis. Thus no industry-wide grove replanting policy can be developed from this model. This analysis showed that replanting HLB-infected groves to advanced production systems (APS) groves gives the grove perpetual life if tree resetting is practiced and an economic life greater than 25 years, if resetting is not practiced. Thus, APS technology offers the potential for growers to economically survive until a cure is found for HLB. We plan to publish preliminary results of this grove replacement model in Citrus Industry Magazine in the spring of 2011. Final results will be developed and published when data on an advanced production system grove are available.
The following transgenic plantlets have been produced in Dr. Grosser’s lab: Beta glucanase with 35-S constitutive promoter: Vernia 15 plants; Valencia 8; OLL orange 5; Carrizo 10; Duncan 2, and Beta glucanase with Suc2 phloem-limited promoter: Vernia 6, Valencia 5; OLL 7; OLL#8 9. All of these plants and some earlier ones are being maintained and/or increased in a greenhouse. Some earlier produced plants have been challenged with the HLB bacteria. Additional plants transformed with Liberibacter virulence genes are being maintained by Dr. Wang’s group. As these plants mature, they will be challenged with Liberibacter bacteria and symptom expression will be monitored. Samples were taken from 131 Poncirus plants at two sites to see if any of these plants are PCR positive for HLB. Carrizo and Swingle plants at one of these sites were positive last year, unlike their symptomless expression when grown in a greenhouse or growth chamber.
During the second period, we accomplished manual verification of structural and functional predictions for Candidatus Liberibacter asiaticus proteins incorporated in KEGG pathways. And as a pilot study for a more in-depth analysis of a crucial subsystem, we carried out a project to classify all the ABC-transporters in Liberibacter. More specifically, in order to understand the function of a protein in the context of the whole organism, we manually analyzed proteins in subsystems or pathways from KEGG. For all proteins included in KEGG (about 500 proteins, half of the genome), we selected the best homologous template for structure modeling, analyzed the domain architecture and predicted the function by combining the evidence from homology, structure prediction, surrounding genes in the genome, and the integrity of each pathway. For 95% of the proteins in these major pathways, we were able to provide a confident structure prediction and most of their functions can be predicted. By comparing with the pathways in close bacteria, e.g. Rhizobium etli, we noticed several inconsistencies within the Liberibacter genome. Some key enzymes appeared missing, and others did not fit in the context of a pathway. The former cases may reveal non-homologous iso-functional enzymes which can be used as possible targets to control the bacterium. On the other hand, we carried out an in-depth study of all ABC-transporters in Liberibacter. Starting with ABC-transporter ATPase, which is a conserved family of proteins with diagnostic sequence and structure motifs, we assembled all the component proteins of ABC-transport system. We attempted to clarify their functions by comparative analysis, classified their structures and studied their evolution. The manuscript describing the results is currently in preparation. Briefly, we found 17 ABC-transporter ATPases in the genome and identified 15 ABC-transport complexes consisting of 39 proteins in total. The two remaining ABC-transporter ATPase homologs apparently do not function as ABC-transporters. One of them was homologous to SufC, which is part of the iron-sulfur cluster assembly complex. The other one (gi|254780233) is homologous to ChvD, which is involved in regulation of virulence gene expression in Agrobacterium tumefaciens. Given its important function in virulence gene expression of a close bacterium, we proposed that this gene should be of interest. Currently, we are trying to expand the manual structure-functional analysis to proteins that are not mapped in KEGG. These proteins usually have fewer homologs available to base our predictions on, so for many cases, ab initio methods to fold proteins in silico will be applied. In addition to ABC transporters, we also will carry out in-depth analyses of other interesting systems in the bacteria, such as other transporters and sugar metabolic pathways. We believe that such an analysis will result in better understanding of the physiology of this bacteria and the mechanisms of its virulence.
1- The growth room construction started on October 22nd, 2010, projected finish date is February 11th, 2011. The construction is already one week behind according to the schedule. They are approximately half way done with the wall insulation, the ceiling insulation has not yet begun. We set up a meeting to discuss disposal of the waste stream for the grow room. The director of UF/IFAS Pesticide Information Office, the coordinator of the UF facilities planning and operations and a representative of the EPA were involved in the discussions. A final list of pesticides and chemicals to be used in the grow room was finalized in order to comply with all environmental regulations. 2 – All in vitro clean shoot tips (Hamlin 1-4-1, Valencia 1-14-19 and Pineapple F-60-3) to establish the mother plants were released from Dr. Peggy Sieburth lab, from the Department of Agriculture, Winter Haven. They are still in test tube conditions. The shoot tips are already 3-month old and they are ready for grafting onto rootstocks grown in pots. Since the growth room is not ready, we have transferred them to fresh medium to keep them alive until our growth room is completed. A second transfer of the shoot tips to fresh media is scheduled for January. As mentioned in earlier reports, this material is needed to be grafted on rootstocks in pots at approximately 2 months of growth. Another factor we are worried about is the current rootstock growth conditions. In the lab where they are developing, the lack of appropriate light, temperature, and space to grow them is jeopardizing 6 months worth of work. They are growing but with extreme difficulty. The initial planting material was discarded since it was getting too old and new batches are already growing but until we don’t have a better place where growing them on clean conditions we will continue to struggle. Even if we can occupy the new growth room the last week of January, we are still going to be concerned about achieving our desirable results. 3 – The growth room technician was finally hired and will start working the first week of January. He will go to Spain for training next Spring. 4 – The lab is 80% set up and, after removal of all plants to the growth room, we will clean and finish setting up the lab for full in vitro culture purposes. Supplies and equipment for the growth room will be purchased once it is completed.
Our research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. During the first year of the grant’s period peptides, proteins and RNAi moieties were tested by feeding them to psyllids using artificial diets. The diet was optimized by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period and we identified suitable buffers and optimal pH. Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from Diaprepes abbreviatus, the citrus root weevil, were found to be excellent candidates; causing high mortalities when fed to psyllids by artifical diet. Ten psyllids genes representing three gene families of cathepsins (five genes), vacuolar ATPases (four genes), and tubulin (one gene) were targeted and their dsRNA (16 ng/’L) fed to psyllids using artificial diets. Three vacuolar ATPases and three cathepsins (B, L and F) showed significantly higher mortality than the controls. In the first quarter of the second year period our studies continued to characterize the cause of increased psyllid mortality induced by feeding of Double-stranded RNA (dsRNA) molecules targeting specific psyllid genes. Large scale experiments were conducted to harvest sufficient RNA for Northern blot characterization of the integrity and abundance of specific psyllid mRNAs that were targeted and showed enhanced insect morality. The Northern blot analyses although cumbersome and time consuming, are essential for complementing Q-RT-PCR based analyses of targeted transcript abundance. To further support and enhance our RNAi research observations using artificial feeding chambers, we developed a detached leaf assay that supports adult and nymph psyllid survival and allows dsRNA uptake into intact citrus leaves on which the psyllid are naturally feeding. Initial results suggest that transcript specific mortality induced by feeding dsRNA to psyllids in artificial diets can be reproduced using the detached citrus leaf assay. The assay was developed to show that low doses of dsRNA circulating within the phloem can shut down key biological genes in psyllids when ingested, and thus support the possibility that RNAi strategies can be developed to control psyllid feeding on citrus and, therefore, control the spread of HLB. As part of this research a dsRNA virus was also discovered in psyllids and was characterized. This virus is present in natural psyllid populations within Florida, but accumulates to higher levels when the psyllids are maintained in greenhouse colonies. Because it is possible that dsRNA viruses can suppress the RNAi machinery of an insect, we are currently developing dsRNA of virus free psyllid colonies to support future RNAi research in psyllids.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. These include Polyamines such as putrecine, spermine and spermidine; and Antioxidants such as lipoic acid, glycine betaine and glutathion. Lipoic acid continues to yield the best results. A carrot suspension culture overlay procedure is also being evaluated. Experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency are also underway, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publication from work on alternative transformation systems: Dutt, M. and J.W. Grosser. 2010. An embryogenic suspension cell culture system for Agrobacterium mediated transformation of citrus. Plant Cell Reports. 29(11): 1251-1260. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ A field trial was established in collaboration with Mr. Orie Lee to evaluate sweet orange seedlings from six selected somaclones of precocious ‘Vernia’ sweet orange under commercial conditions. Juvenile Vernia trees are less thorny than other commercial sweet oranges, and our plan is to girdle the trees to induce early flowering and fruiting once the trees reach adequate size. The goal is to quickly establish a producing grove from juvenile budwood – as necessary to have a system for comparable transgenics. Significant progress was also made to identify rootstocks to enhance early production from juvenile scions, including subsequent transgenics. The 2.5 year old field trial using a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks is showing significant rootstock affects on precocious bearing – the best selections from this trial will be tested with juvenile transgenics, based on yield and fruit quality data to be taken in February. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious ‘Vernia’ sweet orange (including somaclones) were regenerated and successfully micrografted for further study of early flowering and transgene expression. 31 transgenic ‘Vernia’ trees were produced containing four different gene constructs. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
After stabilization in the hydroponics system, greenhouse plants have been subjected to the three levels of boron fertilization (high, normal, and deficiency). As the nutrient levels stabilize to the new fertilization treatments we will inoculate the seedlings with HLB and monitor for Las titer and symptom development. Determining the mechanism of this possible HLB symptom escape will provide important information about how Las causes disease and provide a possible strategy for HLB management by citrus growers.
In cloning the three SA genes, EDS1, SID2, and WIN3, we currently confirmed the cloning of the full-length ctEDS1 and are in the process of moving the sequence to the binary vector for plant transformation. We showed in the last progress report that we obtained 3′ end RACE sequence for ctWIN3 and 5′ end RACE sequence for ctSID2. In order to amplify the other ends of the two genes, we tried to design different primers for RT-PCR. We also performed TAIL PCR, in which we used citrus genomic DNA as a template in a series of PCR in order to obtain the missing regions of the two genes. However, these attempts were unsuccessful. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we have been doing bioinformatics analysis and have identified additional SA genes that have citrus homologs with available sequence. We are currently design primers to further amplify these additional SA genes. For ctEDS5/pBINplusARS transformation, we obtained 5 Col-0 and 5 eds5-1 carrying the transgene. We are in the process of screening T0 seeds for additional independent transformants. In the meantime, we planted these 10 transgenic plants for disease resistance assay with Pseudomonas infection. We continue to characterize the transgenic plants overexpressing ctNDR1/pBINplusARS, ctNPR1/pBINplusARS, or ctPAD4/pBINplusARS. We obtained 4 homozygous ndr1 + ctNDR1/pBINplusARS and performed disease resistance assay. The recent data confirmed our earlier report that ctNDR1 complemented Arabidopsis ndr1 mutant. Additional analysis will be conducted to verify this result and to further characterize the defense phenotypes of the transgenic plants. For plants overexpressing ctNPR1/pBINplusARS or ctPAD4/pBINplusARS, we did not observe complementation of npr1 or pad4 mutant with transgenic plants currently obtained. We reason that overexpression of these two genes may be toxic or citrus cDNA clones may not be well expressed in Arabidopsis. We are currently trying to clone the genomic fragments of these two genes. We will repeat npr1 or pad4 complementation once we obtain the genomic clones.
The main objective of the project is to identify all the microorganisms in the phloem cells of HLB affected citrus trees. To this end, we carried out extensive microscopy imaging of vascular tissues from Candidatus Liberibacter asiaticus (Las) infected citrus and periwinkle plants. The following are conclusions from our investigation. 1) Las is the sole type of bacterium morphologically identified in the phloem of Las infected (Las+) citrus plants. Neither mollicutes (bacteria without cell wall) nor flagellated bacterial cells were detected. Occasionally, Citrus tristeza virus was seen in companion cell nuclei in the Las+ phloem of citrus plants. 2) Phage particles were observed in Las+ periwinkle phloem cells. The phage particles were detected only in Las+ periwinkle plants and they appear to lyse Las cells. Based on their structural features, the phage particles seem to be a member of Podoviridae. To the contrary, no phage particles were identified in Las+ citrus phloem cells. 3) In the phloem cells of Las+ infected leaves, excessive amounts of callose accumulate around the plasmodesmata (PD) and sieve pores. 4) The leaf chlorosis symptom of HLB is due to starch over-accumulation in the leaf chloroplasts and callose deposition in the phloem takes place prior to leaf chlorosis. 5) Rate of photoassimilate export from leaves is reduced in Las+ plants. These results provide evidence that Las is responsible for HLB symptoms and that callose formation around PD and sieve pores of Las+ plants could disrupt phloem transport. The low titers of Las and inactivation of symplastic transport in HLB infected trees suggest that the HLB symptoms are self-inflicted by the plant’s response to Las infection rather than caused by direct injury to the phloem by Las cells or by physical occlusion of the phloem by bacterial proliferation. We have submitted a manuscript to Phytopathology reporting conclusions 1, 3, 4, and 5. Conclusion 2 will be published in a manuscript from Dr. Dean Gabriel’s lab.
During the 2nd quarter of funding, the Core Citrus Transformation Facility (CREC) continued it’s mission of producing transgenic Citrus plants according to the orders from multiple clients. The demand for genetically transformed citrus plants remains high. Most recently, CCTF received three new orders to produce transgenic grapefruit carrying genes harbored in following vectors: p19-5; p20-7; and p21-1. The bulk of the work presently revolves around orders placed in the previous quarter but work also goes on to complete older orders. Out of presently serviced orders, all except two are concerned with resistance of different citrus cultivars to diseases, primarily HLB and canker. The following transgenic citrus plants were delivered to various researchers: Resistance to bacterial diseases-canker and HLB: 1) N1* gene: two Duncan plants; 2) pCIT108P3 vector: two Flame plants; 3) NPR1: three Flame plants and superNPR1-four Hamlin plants; 4) AS7 gene: eight Duncan plants and A13* gene: four Duncan plants; 5) pMOG800 vector: two Duncan plants. Resistance to CTV: 1) Gene in p33 vector: 18 Mexican limes, 16 C. macrophylla, and seven Hamlin plants. Orders not associated with citrus disease resistance: 1) CL1 gene: one Duncan. 2) pHK vector: 12 Mexican limes. During this quarter, more than forty recovered new transgenic plants were soil-adapted, and are ready for PCR testing to confirm the presence of the trasngene of interest. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
The objectives of the first year’s research project are focusing on: 1. Conduct genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from genomic sequences of ‘Candidatus Liberibacter asiaticus’ (Las). Design and develop PCR-based multi-locus SSR molecular diagnosis assay. 2. Analyzing Las population structures, assessing the genetic diversity of Las in Florida populations. We assessed Florida Las population and compared it with a global genetic diversity of Las populations using a multi-locus SSR marker system. A total 293 HLB samples used for population analysis were composed of isolates obtained from the US (177 isolates), China (42 isolates), India (35 isolates), Brazil (22 isolates), Cambodia (11 isolates), Vietnam (3 isolates), Taiwan (1 isolate), Thailand (1 isolate), and Japan (1 isolate). This sample population had an abundance of sequence types, consistent with the sampling diversity and the type of marker (SSR) used for this study; a total of 147 sequence types (STs) were identified from the 293-member sample pool (Supplemental Table 1). All of the STs identified within the sample population were restricted to the boundaries of their country of origin; in other words, we found no individual ST in more than one country. Profile frequencies and allele frequencies were calculated for this sample population. Based on allele frequencies of SSR loci among these populations, a genetic similarity matrix that consisted of 71 alleles and 293 samples was generated. The genetic distance analyses were performed using Neil’s coefficient with 1,000 bootstrap and 95% confidential intervals. A pair wise population genetic analysis indicated that among four Las populations, the genetic distance between Florida vs China Las populations is 0.61, while the genetic distance between Florida vs India is 1.35, Florida vs Brazil is 0.96, China vs Brazil is 0.84, India vs Brazil is 0.95 and China vs India is 1.72, respectively. The results indicated that the genetic distance between Florida and China have the closest distance (0.61) as compared with other populations. Analysis of genotypic profiles present amongst an HLB sample population allowed us to make inferences about the relationships between L. asiaticus isolates associated with HLB. From this work, we conclude that at least two major clonal complexes of L. asiaticus are associated with HLB worldwide and that the recent introduction of HLB into the southeastern US probably occurred as a result of at least two separate events. The panel of markers we introduce here will be useful for future population studies of HLB and may also aid in the identification of segments of the L. asiaticus chromosome associated with bacterial virulence. High percentage of commonly-shared allele types detected in both populations suggests that Las populations in Florida and China are more related. This leads to a hypothesis that Las populations in Florida could be possibly derived from China. It is not clear if the introduction was from single or multiple times and/or from multiple sources as well. Our results demonstrate the useful of multi-locus SSR marker system for genetic analysis of Las. Bacterium has very compact genome. Sequencing variations of SSR loci located within or near the coding and/or gene regulatory regions could have profound effect on gene expression and functions. We have mapped SSR loci genome containing genes of interest. Our next step is to examine the functionality of the candidate genes using a standard in vitro heterologous expression system. This type of study will facilitate the link of DNA-based genotyping to phenotyping of Las. We have accomplished objective 1 and 2 during the first year of the project. Research had been summarized and reported in 2009 and 2010 American Phytopathological Society meetings.
We want to obtain a pure culture of Ca. Liberibacter asiaticus (LAS) by first co-culturing the bacteria with insect cells. The strategy consists of primocultures of the bacteria in insect cell cultures used as feeder cells. For this annual report our project objectives are described and discussed below. Objective 1: LAS inoculum. LAS source materials are from infected symptomatic citrus trees from Vietnam. Transmissions from citrus to citrus or from citrus to periwinkles are performed via the insect psyllid Diaphorina citri and subsequent grafting. A maceration method was found to be the most appropriate way to release the bacteria in the insect cell cultures. Antibiotics are used to select for Gram negative bacteria. Objective 2: Primo-cultures. Various insect cell cultures in various culture media were tested. -We didn’t detect any Las in Mamestra (hemocyte, ovarian cells) or Spodoptera (hemocyte cells) cell lines after inoculation. -We detected LAS in two lines of drosophila cell cultures by direct PCR after inoculation. One line lost the detection after 6 subcultures. For the second line, positive PCR signal was obtained up to the 20th subculture, then LAS detection troubles occured due to high insect cell density and we detected the presence of another bacteria (Bradyrhizobium). We started new LAS primo-cultures with much less drosophila cells and got LAS positive cultures for some combinations of cell line/culture medium, still positive after 5 weeks and successive dilutions (confirmed by sequencing). -First attempt to co-culture LAS with an Aedes albopictus insect cell line failed. However, adding an antioxidant and osmoprotectant at the time of inoculation allowed us to get LAS positive Aedes cultures still positive after 9 weeks and successive dilutions (confirmed by sequencing). We are currently adapting conditions of described LAS qPCR detection to quantify LAS in insect cell cultures. We can detect LAS bacteria by qPCR but more efforts are needed to quantify bacteria:insect cell ratio with a multiplexed qPCR approach. Our objectives 3 and 4 are aiming to improve culture conditions to get a higher bacterial titer and to free the co-cultures of insect cells (axenization). Objective 3: Axenization. LAS/Aedes primo-cultures were obtained at a low insect cell concentration. We are progressively diluting the concentration of insect cells through each new passage . Objective 4: Medium optimization. To maintain the bacteria for a longer period of time and to reach higher bacterial concentration, we started complementing the primo-cultures with various sugars, vitamins described in citrus/periwinkle phloem. In parallel we analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to define limiting factors and/or growth inhibitors. Of the complements tested we selected sodium pyruvate, proline and fructose for their positive effect on the bacteria detection and they now are systematically assayed on our new inoculations. We are analyzing sugar, amino-acids and minerals and trace elements variations in insect cell culture media over culture time to identify potential LAS growth limiting factors. We are reaching our milestones for the first year of this project with significant progress on objectives 1, and 2 (inoculums, primo-cultures). For objective 2, we will test new insect cell lines. As at least two laboratories involved in the FCPRAC program established Diaphorina citri cell lines, we will request for these lines to trial our inoculation protocol and try to obtain LAS/ D. citri co-cultures. We are now fully involved in objectives 3 and 4 (axenization/medium improvement). If they succeed in time, we will attempt to inoculate cultivated bacteria through Diaphorina citri to Citrus trees (objective 5).
Physiological changes in HLB fruit: Global gene expression in HLB-infected fruit tissues was evaluated using an Affymetrix array containing 30,279 Citrus spp. ESTs. Flavedo (FF), vascular tissue (VT), and juice vesicle (JV) tissue of symptomatic (SY), asymptomatic (AS) and healthy (H) fruit harvested from sweet orange ‘Hamlin’ and ‘Valencia’ cultivars was investigated. Transcriptional profiles indicated the number of genes impacted by HLB was highest in FF and VT and least in JV. In ‘Hamlin’, over 860 genes were changed in SYVT and SYFF compared with H tissues. In ‘Valencia’, fewer genes were changed in SYVT and SYFF, but approximately 50% additional genes (397 genes) were changed in SYJV compared with ‘Hamlin’. As in SY tissues, ASVT and ASFF were strongly impacted and had similar gene changes in ‘Valencia’. However, only 28 genes were altered in ASJV compared with HJV. The top five gene groups with high numbers of changes included transporters, carbohydrate metabolism, genetic information processes, phytohormone metabolism and defense responses. Highest titer of the HLB bacterium was found specifically located in SYVT. Gene changes in SYVT were associated with host-pathogen interaction and seed abortion, and those in SYJV with fruit size, juice sac maturity and morphology, and juice quality. Similar to SY fruit, girdled fruit were small and could abscise prematurely. Comparison of gene expression between HLB-infected and girdled treatments is ongoing to differentiate HLB-mediated gene changes from those of girdling-related carbohydrate loss. We studied carbohydrate and phytohormone changes in HLB affected fruit to explain symptom development since 1) carbohydrate shortage has been linked to fruit growth arrest and eventually abscission and 2) hormonal signals regulate, at least partially, fruit set and development. SY, AS and H fruit were harvested from ‘Valencia’ trees (Citrus sinensis L. Osbeck) infected (SY and AS) or not (H) with the HLB pathogen, as verified by PCR. Mature SY fruit weighed less, had lower ‘Brix, were smaller, had more aborted seeds, and were greener than AS or H fruit. Starch and sucrose contents were lower in SY mature fruit flavedo compared with H and AS. SY and AS fruit harvested 7 and 12 months after full bloom produced significantly less ethylene than H fruit. Indole-3-acetic acid (IAA) and abscisic acid (ABA) contents in flavedo removed from the stylar-end, middle section, or stem-end of fruit, generally were higher in SY fruit than in AS and H. Flavedo excised from the misshapen area of SY fruit had significantly higher IAA content when compared with the normal-sized area of the same fruit on the opposite side. This increase corresponded to an increase in hypodermal cell area in S fruit flavedo. Overall, these data reveal an imbalance of carbohydrate and hormonal status in fruit from HLB-infected trees and suggest a role of such changes in fruit symptom development. Extension and education: The effects on Huanglongbing on Florida Orange Juice brochure by Danyluk, Spann, Rouseff, Goodrich Schneider, and Sims has been submitted to EDIS for publication and was distributed to all attendees at the 2010 International Citrus and Beverage Conference (ICBC) in Clearwater Beach, Florida. An educational display was also created and displayed at the 2010 ICBC meeting. ICBC is an annual meeting with an average attendance of 350 individuals from the citrus industry.
n FL nurseries, rootstock seed trees are located outdoors and only protected from psyllid transmission of Candidatus Liberibacter asiaticus (Las) by insecticide applications. In 2008, a survey detected two Carrizo citrange trees as HLB+. Given the potential risk for seed transmission and introduction of Las into nurseries by seed from source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008 seed were collected from mature Pineapple sweet orange trees in Collier Co. and in 2009 from Murcott tangor trees in Hendry Co., FL. For Pineapple orange, 415, 723 and 439 seedlings and for Murcott, 332 seedlings were tested at least twice by qPCR using 16S primers. In 2007, a single Pineapple seedling was suspect HLB+ but upon repeated testing was negative. From nurseries in 2008, 290 seedlings were recovered from fruit located on symptomatic branches of 2 Carrizo trees, and in 2009, 125 seedlings were recovered from 2 trees of Swingle citrumelo, 649 from 4 trees of ‘Kuharske’ Carrizo, 100 from 1 tree of Cleopatra mandarin and 100 from 1 tree of Sekwasha mandarin. In 2008, one suspect HLB+ Carrizo seedling was detected but HLB+ status was not confirmed after repeated testing. In 2009, a single questionable PCR detection for Cleopatra mandarin was obtained. Subsequent detection occurred in only 75 and 33% of repeated 16S runs from two DNA extractions and the assay was negative using beta-operon primers. Despite the occasional HLB+ test results, no plants have ever developed HLB symptoms and repeated testing has never confirmed anything other than the transient presence of Las in seedlings grown from seed obtained from Las-infected trees.
Objective 1 (confirm biofilm formation by X. citri subsp. citri in comparison with other bacteria that are well known to form biofilms). Using stable and labile Gfp expressing strains, aggregates (biofilms) were confirmed to be formed by viable cells. Xcc-A is the wide host range strain attacking most citrus cultivars, whereas Xcc-Aw is restricted to Mexican lime and Xac to Swingle citrumelo. The three strains aggregated on the leaf surface of Swingle citrumelo and Mexican lime. However, higher aggregation on the leaf surface was observed for Xcc-A than for Aw or Xac leaf. In addition, higher aggregation by Xcc-A developed on Mexican lime than on Swingle citrumelo. Xcc-A formed large complex aggregates on these hosts whereas Aw or Xac formed filaments. A similar relationship among strains and hosts was observed for biofilm formation on fruit. Xcc-A strain was able to aggregate and form biofilm on the surface of both Mexican lime and grapefruit, but Aw produced biofilm only on Mexican lime and none on grapefruit. Xac did not form biofilm on either grapefruit or Mexican lime. Based on differential aggregation among these strains on compatible and incompatible hosts, biofilm formation on the plant surface may play a role in the disease causation process. What role biofilm formation has in the infection process is still unknown. However, we hypothesize that Xcc-A strains may be more virulent in part because they are a better colonizer of compatible hosts than less virulent Aw and Xac. gumD is involved in biofilm formation. A gumD transcript of 445 bp is only amplified from active cells, therefore its presence may be used as a viability marker. gumD transcript in Xcc-A was higher in cells 72 h after biofilm formation (aggregated state) than in cells in suspension (planktonic state) signifying higher cell activity. rpf gene is expressed during quorum sensing in the biofilm formation process. rpf is involved in synthesis of a DSF (diffusible signal factor) related to quorum sensing and is expressed at a higher level in Xcc-A during biofilm formation. Other studies are currently in progress using genes expressed during chemotaxis (cheZ, motA, fleN), and cellular division (Rsp, SulA) to evaluate their role in the Xcc-A colonization process. Objective 2. A wide host strain of Xcc-A with stable and labile Gfp was grown in the presence of different concentrations of the bactericidal compounds, CuSO4, NaCl and SOPP. The treated bacteria were then sprayed on Swingle citrumelo leaves and biofilm formation on the surface observed at different days after inoculation (dpi). At 1 dpi, aggregates were detected in all the treatments but a larger area of aggregation occurred after Xcc-A was exposed to NaCl. After SOPP exposure, aggregates were restricted to certain locations on the leaf surface proximal to stomata and in depressions. At 3 dpi, NaCl treatment showed extensive and thick biofilm formation while, in the case of SOPP, living bacteria were mainly found inside in the sub-stomatal chamber and in the apoplast. At 9 dpi SOPP biofilm on the leaf surface was almost non-existent although a substantial bacterial population was present in the sub-stomatal chamber and apoplast. In summary all bactericides do not reduce the bacteria’s ability to form biofilms i.e. neither NaCl nor SOPP prevented bacterial infection.
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