The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ Baseline sensitivity of Alternaria alternata population to boscalid experiments were finalized and analyzed. ‘ Statistical analysis of QoI-fitness and baseline sensitivity to boscalid was finished. ‘ A subset of 15 isolates was selected to molecularly characterize the structure of the iron-sulfur (SDHB) and membrane-anchored (SDHC and SDHD) subunits of the succinate dehydrogenase (SDH) of A. alternata. ‘ DNA extraction was performed using the 15 isolates previously selected. ‘ Nine pairs of primers were developed to amplify different segments of SDH’s gene. ‘ Three pairs of primers: AASDHBF2/AASDHBR3, AASDHCF1/AASDHCR1 and AASDHDF1/AASDHDR1 were selected for further analysis. ‘ DNA cloning and sequence for 15 isolates using three pairs of primers were performed. ‘ DNA assembling for individual isolates within corresponding SDH gene was done. ‘ Using the sequence information, the molecular characterization of SDHB, SDHC and SDHC subunits were established. ‘ The paper ‘Stability and fitness of Alternaria alternata tangerine pathotype’ is on-going. ‘ For the project: ‘Detection of resistance in the non-pathogenic Alternaria population in tangerine groves’, a new set of primers were designed (three pair of primers). ‘ The primers CytbF1/CytbR1 were selected to clone and sequence the partial segment of cytochrome b using six selected isolates. ‘ RFLP-PCR analyses were performed in a subset of 20 isolates of non-pathogenic A. alternata to identify the point mutation G143A.
Based on previous results (see previous reports), we designed a model to test the effectiveness of the selected chemicals. Shoots were collected from a single HLB-symptomatic Valencia Orange (C. sinensis) tree, infected with ‘Ca. L. asiaticus’. Previous studies have reported greater numbers of viable ‘Ca. L. asiaticus’ cells in the sieve elements of young, asymptomatic leaves, collected from new flush. All leaves used for this study were collected from new flushes on highly symptomatic branches. Nine leaves were collected for each treatment and control group. Samples were then incubated for 6 or 24 h (with or without chemical). We followed the transcriptional activity of the 16S RNA gene and the L10 ribosomal protein (encoded by the rplJ gene) as viability parameters. The amplification values were normalized to the plant gene cox2 and are expressed relative to the control (incubated without chemical) samples. After 24 h of incubation, significant differences were observed in samples treated with small molecules. Expression of the 16S RNA gene was repressed in the treated samples. The effect of the selected chemicals on the expression of the specific genes ldtR and ldtP was then determined in the infected leaves. The expression values are calculated relative to the 16S RNA gene, to assess the specificity of the chemicals to target genes. Compound A showed a strong effect (9.0 .fold decrease) on the expression of ldtR after 6 h of incubation, while B displayed similar repression values after 6 or 24 h (5.1 .and 5.2 fold, respectively. The expression of ldtP showed constant and incremental repression values over time. Compounds B and C reached maximal values of 16 and 17, respectively, while A showed a maximal repression of 7.0 fold. These results indicate that the small molecules tested act specifically on the ldtR activator. We hypothesize that in ‘Ca. L asiaticus’, expression of LdtP is increased in response to biotic stress, allowing persistence of the bacteria within the phloem of the tree. As such, the regulation of ldtP expression through inactivation of LdtR with small molecules represents a direct means of influencing stress tolerance, and survival of ‘Ca. L asiaticus’ within the host. These results demonstrate that the rational selection of chemicals to inhibit specific targets is effective to design new treatments to diminish Liberibacter asiaticus viability on infected trees. The next logical step of our research is the treatment of infected trees directly on the field to evaluate its long term effectiveness.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the LasA1 gene, the N-terminal region (LasA1-A), two version for the repeat region with different number of the repeat sequences (LasA1-B0 and LasA1-B1), the C-terminal region (LasA1-C), and the full LasA1 gene. We are analyzing these constructs for their transient expression in Nicotiana benthamana and stable expression in transgenic Arabidopsis thaliana. The transgenic lines were obtained by floral-dip transformation of Arabidopsis Col-0 plants and we are currently verifying the gene insertion and mRNA expression level on T2 Arabidopsis. Three transgenic T3 lines expressing the gene are selected for analyzing phenotypes and protein localization using GFP pGWB2 vector. We are testing the expression level of the gene constructs that were transiently expressed in N. benthamiana with 35S, PFLAG and GFP pGWB2, 6 and 12 vectors. Different constructs of LasA1 and LasA2 proteins using GFP vectors for localization, and using the PFLAG vector for protein-protein interactions were made, respectively. pGWB-PFLAG c-terminal and n-terminal Autransporter plasmids: LasA1, LasA2 and LasA1-a/b/c were over-expressed in Nicotiana benthamiana under 35S promoter via agrobacterium-mediated transient expression. Inoculated leaf samples were collected after 3 and 6 days post inoculation (dpi) and stored at -80 C for further analysis. Immunoprecipitation and elution of FLAG-tagged autotransporters, as well as ATP quantification of the challenged samples are underway.
This project is to assess how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. Our main accomplishments on this project are listed below. 1) Electron microscopy examination of the sites on the leaves of citrus plants where HLB-positive psyllids fed for 7 days demonstrated that even at early stages of infection (starting from 3-4 weeks after the beginning of the experiment) the bacteria could be already visualized in the initial sites of introduction. 2) In order to characterize inoculum sources of the bacterium available for psyllids within an infected tree, we examined the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes, young growing or matured symptomatic ones. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. We also conducted a similar experiment that was slightly modified in a way that psyllids fed on old and young leaves that were detached from plants and kept in 50 ml tubes (‘detached leaf experiment’). Some differences in the bacterium acquisition were obtained from these two experiment series. On average 48.33% of psyllids fed on old symptomatic flushes tested positive and 58.33% of psyllids fed on young pre-symptomatic flushes were positive. In the ‘detached leaf’ experiment, an average acquisition from young pre-symptomatic tissue was significantly higher than from old symptomatic flushes: with average of 64.26% and 23.9%, respectively. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. Analysis of numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes revealed that more receptor plants that were inoculated by psyllids kept on young flushes became infected (52% of Duncan grapefruit plants and 53% of Madam Vinous sweet orange plants) and less proportion of receptor plants inoculated with psyllids that fed on old mature flushes got infected (19 and 33% of the same varieties, respectively). 3) In order to assess what types of flushes are more susceptible to psyllid inoculation with the HLB bacteria, we exposed sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes to HLB-infected psyllids (“no young flush” plants). According to our data, both young and mature flushes could be inoculated by psyllids, yet inoculation efficiency of mature flushes is significantly lower. 4) Overall, our results support the initial observation of young flushes being more likely crucial for the disease spread at both steps of the pathogen transmission, either acquisition and inoculation are higher when young flush are present. Nonetheless, transmission associated with old tissues, which occurs at a reduced level, should not be ignored also. 5) To examine psyllid transmission rates to different citrus genotypes, we analyzed psyllid inoculation of 6 different varieties of citrus: Valencia sweet orange, Duncan grapefruit, Persian lime, Eureka lemon, Carrizo citrange, and Poncirus trifoliata. Those varieties represent plants with different degrees of susceptibility to HLB. The first four varieties showed the highest infection rates (80-100% infection), while only about 10% of Carrizo citrange and Poncirus trifoliate became infected. Poncirus and poncirus hybrids have been shown to have much greater tolerance to HLB. The fact that they are also more tolerant to psyllid inoculation with the bacterium suggests that developing hybrids of such varieties that in addition to being more tolerant would also have acceptable horticultural characteristics and would produce fruit and juice of a sufficient quality could be a solution to battle HLB epidemics before more sustainable approaches are in place.
To determine the effect of azelaic acid in the defense response of citrus we pre-treated susceptible ‘Duncan’ grapefruit plants with this chemical via leaf infiltration and subsequently inoculated the infiltrated portion with Xanthomonas citri pv citri (Xcc). Because this pathogen can be infiltrated at a specific time, location and concentration, compared to the graft inoculations necessary to transmit Candidatus Liberibacter asiaticus, we thought it would be a quicker way to determine if this compound has any effect on resistance. After Xcc inculcation the population growth was determined using colony forming units (cfu) using a standard procedure. There was no effect of azelaic acid in the Xcc growth when compared to water control. In a separate set of experiments we have initiated an Illumina NextGen DNA sequencing project comparing ‘Sun Chu Sha’ mandarin (HLB tolerant) and ‘Duncan’ grapefruit (HLB sensitive) infiltrated with Candidatus Liberibacter asiaticus flagellin 22 (CLas-flg22) peptide and water control. The idea is to identify genes that may be part of the PAMP-triggered immunity (PTI) and differentially expressed in the distinct genotypes and treatments. This is underway and we expect to be analyzing the data in the next quarter.
A new FT construct,FMVcDNA27, containing an FT3 cDNA insert in the pCAMBIA 2201 vector has been made with a constitutive FMV promoter that proves to be as effective at transforming citrus and tobacco as the corresponding genomic construct that has been previously used. The FMVcDNA27 construct will be used to develop a chemically inducible system for the expression of this transgene. The inducible promoter systems from the Danforth Foundation mentioned in the previous quarterly report update was not used due to unforeseen issues; therefore the work will be continued using a transcription activator-like (TAL) effector system inducible by methoxyfenozide that will activate the naturally present FT3 gene in citrus. This promoter will utilize chemical-inducible ecdysone receptor-based expression. Research has also been conducted that looks into other endogenous plant chemically inducible promoters that are not turned on by chemicals in the media used to transform plants for use in controlling the FT3 gene expression. A manuscript comparing the behavior of the three genomic clones from citrus when overexpressed in tobacco has been completed and is undergoing further review before publication. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype has been completed and gene expression levels have been compared in a month-to-month basis using the comparative CT method from qualitative Real Time PCR. The results show a promising patter that could potentially clarify the flowering pathway and the physiological effects of these three genes as it relates to the induction of flowering. The data is being currently analyzed for statistical significance and will be cross-examined using a higher concentration of DNA to verify the results. SDS pages and western blots have been done with the synthesized FT3 protein in order to identify if the HA tag and antibody will be an effective method to test for the presence of the protein when applied to plant tissue. The synthesized protein showed high specificity the HA-antibody and therefore this method will be used to assess the presence of the FT3 synthetic protein in further studies. The same procedure was performed with the Arabidopsis FT antibody and endogenous FT in citrus, tobacco and Arabidopsis as to determine if this will be an appropriate assay for the detection of FT3 synthesized protein. Another experiment involving FT3 transgenic tobacco and the effect of plant hormones Ethylene and Gibberellin (GA) as well as a GA pathway inhibitor chemical Paclobutrazol is underway to determine an effective way of preventing precautious flowering of citrus FT3 transgenic plants in tissue culture stages.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the LasA1 and LasA2 genes. We are analyzing these constructs for their transient expression in Nicotiana benthamiana and stable expression in transgenic Arabidopsis thaliana. We have obtained transgenic lines with these constructs via floral-dip transformation. these transgenic lines were verified by PCR and RT-PCR and their segregation in T2 were analyzed. Intriguingly, no obvious abnormal phenotypes were observed in the transgenic T2 lines that over-expressed the LasA1 and LasA2 genes when the LasA1 and LasA2 were fused with GFP at C-terminal. However, transgenic Arabidopsis T2 lines expressing LasA1 or LasA2 with PFLAG showed abnormal phenotypes. Arabidopsis expressing LasA1-PFLAG showed a retarded growth and overgrowth of their roots. Moreover, the leaves displayed different shape with white-silver dechlorophyllation compared to the the wild type, while Arabidopsis lines expressing LasA2-PFLAG showed similar abnormal phenotype with less severity but normal root growth. These results indicated the LasA1-GFP fusion may not function, which may be resulted from the fusion with GFP. We also expressed LasA1 protein using The Champion’ pET Expression System containing a polyhistidine (6xHis) tag in E coli. Purified LasA1 protein will be used for antibody production and crystallization study. In addition, we have made several constructs for development of transgenic citrus via Agrobacterium-mediated transformation. Immunoprecipitation and elution of FLAG-tagged autotransporters, as well as ATP quantification of the challenged samples 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. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. 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 1/16th 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 were planted this quarter, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants with citGRP1 are growing and will soon be tested. Forty of them were test by PCR and twenty two of them are transgenic plants with citGRP1 insertion. RNA was isolated from some of them and RT-PCR showed gene expression. More than thirty kan resistant shoots were obtained from citGRP1 transformed Hamilin. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil and are ready for PCR test. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. Seven transgenic Hamlin with MADS6 were planted in soil. In addition, numerous putative transformants are present on the selective media transformed with different constructs. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many kanamycin resistant transformants were generated on the selective media. About twenty kanamycin resistant shoots are rooted in-vitro and one Hamlin transformant is in soil. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. The construct pBinARSplus:nbFLS2 was used to transform Hamlin and Carrizo. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted in rooting medium and eighty Carrizo and ten Hamlin transformants were plant in soil. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
In this project we are working to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector as a tool to be used in the field to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. Several sets of experiments in which we are examining how preexisting infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees are ongoing. We are also examining the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. We first graft-inoculated sweet orange trees with the T36,T30 or T68 isolate of CTV, the isolates that were propagated in our greenhouse, as well as with CTV-infected material obtained from field (FS series isolates). We are using isolates that contain only single strains and isolates that contain mixtures of strains for primary inoculations. Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. Additionally, real time PCR quantification is also being employed to these tests. Additionally, various rootstock/scion combinations are prepared to be tested in order to find combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the foreign protein of interest from this vector. These combinations include trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. The plants will be used for the experiments similar to the experiments described above.
Function of individual X. citri transcription activator like effectors (TALEs): – Xanthomonas citri strain 2090 from Florida does not contain a typical 17.5 RVD TALE essential for typical pustule formation, but does contain pthA3 15.5 RVD. We cloned this gene and expressed in Xanthomonas citri subsp. citri 306 ‘ pthA1-4 and tested transiently with the 14 EBE promoter:GUS construct in grapefruit leaves. Activity was very low for this TALE, suggesting it may play a marginal role. – Xanthomonas citri strain #93 carries two TALEs – pthC 14.5 RVD and pthC2 17.5 RVD. These were tested in the transient assay and found to weakly activate the 14 EBE promoter:GUS construct in grapefruit leaves. We used an in silico tool – Talvez (http://bioinfo.mpl.ird.fr/cgi-bin/talvez/talvez.cgi, P’rez-Quintero et al., 2013) to scan citrus genomes for TAL effector binding sites, which suggests that PthC2 has differential ability to activate host genes compared to PthC. Transformation: Whereas we observed the expected behavior of gene constructs in transient assays, we have been unable to isolate stable transgenic citrus lines with functional gene constructs. We continue to explore multiple approaches to overcome this technical issue: 1. We tested Carrizo for transformation with the 4 EBE promoter:avrGf1 and challenged with Xanthomonas citri subsp. citri strain 306 and 306 transconjugant carrying avrGf1 into young leaves. The carrizo genotype gave significant resistance for transconjugant Xcc 306: avrGf1 with pathogenicity test infiltrated at 10-3 cfu/ml bacterial suspension. 2. The 14EBE promoter construct efficiency is being tested in tomato; binary vector was engineered with NosT: ProBs314EBE :avrBs4: NosT in T-DNA. AvrBs4 when expressed in tomato results in hypersensitive reaction. The Binary construct in agrobacterium strain Agl-1 was used for transforming tomato Bonny Best and large Red Cherry varieties. Transformant screening is in progress for assessing resistance to bacterial spot disease. 3. We sent two of our constructs for testing in parallel at a contract transformation lab The facility at UC Davis compared the 14 EBE:GUS and 14 EBE:AvrG1 constructs with their standard control plasmid in both Carrizo and tobacco. They found that both of our constructs gave a far lower transformation efficiency than their standard in both tobacco and citrus, and transformants with GUS recovered so far have not showed GUS activity. These results indicate that a likely source of the difficulty we have encountered is the vector. 4. To date we have prepared four promoter constructs in another vector that we use commonly and have good success with. The first set of these transformants are in soil, and we will be able to test the integrity of the inserts by PCR in a few weeks. Preliminary histochemical screening for successful transformation in putatively transgenic shoots of grapefruit, sweet orange and Carrizo showed that transformation has been successful and that transformation was significantly higher in Carrizo than in grapefruit or sweet orange.
In the previous quarter report, the initial development of formulations for two naturally occurring terpenoid essential oils A and B was presented. Oil-in-water microemulsions of the two essential oils (EO) were developed with droplet sizes varying between approx. 5 to 30 nm and upto 14 % (w/w) oils loading. The formulation development was continued in this reporting period and several formulations were prepared from two different oils (EO A and EO B) using agriculturally approved surfactants. Various co-surfactants were employed to increase the loading of the EOs in the formulations. Stable microemulsions were developed for EO B with upto 35% (w/w) oil loading with the aid of co-surfactants. The droplet size distributions for the developed formulations were found to be within the 5 nm to 30 nm range as measured by Dynamic Light Scattering. Initial experiments were also performed to change the droplet charge in the formulations by the addition of anionic / cationic surfactants co-surfactants while maintaining microemulsion stability. The biocidal efficacy of formulations against the surrogate bacteria, Liberibacter crescens were tested in Dr. Triplett’s labs (Microbiology and Cell Science Department). Formulations of both EO A and EO B and corresponding control samples were prepared for testing antibacterial efficacy. Select formulations were tested for the biocidal assay against L. crescens with appropriate antibiotic and media controls. Of the formulations tested, 11 showed bacterial inhibition of over 80% and two showed more than 90% inhibition ( one each for EO A and EO B). Further experiments are being planned to test more formulations and increase biocidal inhibition. Select formulations were tested for phytotoxicity and leaf/ tissue damage in Dr. Albrigo’s labs (Department of Horticulture, Lake Alfred). The two EOs, when applied undiluted, were very phytotoxic. Most of the soft nanoparticle formulations showed low phytotoxicity at 20 to 1 dilutions. Further experiments are being planned for minimizing the leaf/ tissue damage by varying the surfactants/co-surfactants/EO in the formulations. For the application of water soluble antimicrobial agents, preliminary experiments have been performed to produce double emulsions such as water-in-oil-in-water systems. Double emulsions allow for the encapsulation and delivery of water soluble antimicrobial agents for killing the bacteria. The characterization of these emulsions by optical microscopy, droplet size, stability and release of actives is under investigation. Future studies involve improving formulations to provide higher biocidal activity, investigating the uptake of the selected formulations on HLB infected citrus plants, their interaction with the bacteria, Ca Liberibacter asiaticus, as well as their phytotoxicity on the leaves and stems of citrus plants.
Current status of the research: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to develop an efficient system of screening EFR mutants for their binding to elf18-CLas, and in vitro binding system was developed which could be used to screen a mutant phage display library. We demonstrated in vitro binding of both elf18-WT and elf18-CLas to fragments of EFR ectodomain, but not to the ones of related receptor FLS2. Binding of elf18-CLas was weaker than that of elf18-WT, but it was considered that improvements in binding achieved by phage display screening may not be evident considering the binding of WT EFR to elf18-CLas. In addition, recent structural information about FLS2 binding to flg22 indicates the involvement of BAK1 as a co-receptor directly binding the ligand. As the phage display system would not account for this interaction, it may yield mutants which would perturb the binding of BAK1. An alternative system based on split ubiquitin is currently in the process of being investigated, which will hopefully overcome these issues. Additional experiments have also been performed to determine which portion of elf18-CLas is non-functional, by making chimeric elf18 peptides with WT and CLas portions. Both WT-CLas and CLas-WT peptides fail to elicit ROS, indicating there are multiple issues with the function of the elf18-CLas peptide, and thus requiring further investigation. A collaboration is currently being set up with the laboratory of Prof. Chai at Tsinghua to obtain structural information surrounding elf18-WT and elf18-CLas binding to EFR, which would enable more straight-forward testable hypotheses. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Vectors are currently being constructed in the pCAMBIA backbone, under the expression of the 35S/FMV promoter. These constructs will contain: EFR; XA21; both EFR and XA21; and EFR and XA21:EFR chimeric. Cloning of these constructs should be completed in the next few weeks and will then be passed on to the Moore laboratory for transformation in citrus.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 July 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 August. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. A total of 939 psyllids have been found on sticky cards of which greater than 60% are in no-mulch plots with the majority in plots that do not have chemical control rather than plots that receive insecticides. Thus far 35,931 young shoots have been inspected of which 2,061 were infested with ACP. Flush infested with ACP eggs and nymphs have become much more common. Infested flush predominates with 65% found on trees in no-mulch plots. Few (5%) infested flush were seen in plots receiving insecticides with only 0.2% (5 shoots) of total infested shoots found in trees on metalized mulch and treated with soil applied insecticides. Thus, reflective mulch is providing an important added degree of protection against ACP. The third set of leaf samples for PCR analysis was collected 9 July 2013. A single HLB positive tree was detected which had been receiving receiving foliar nutrition but no insecticide in a bare ground plot. The next leaf sample for HLB is scheduled to be collected early January 2014. The third round of tree growth measurements were taken on 12 July. Two trunk diameters are measured as well as canopy height. These measurements revealed larger trunk cross sectional areas in all mulched treatments compared to their no mulch counter parts. Trees receiving insecticide treatments were larger than trees without. No consistent treatment effects differences in tree height were observed. Foliar nutrition is applied during the first week of every month and will continue until the dormant season begins. However, no response in tree growth has been detected from these treatments have thus far. Leaf samples for nutrient analysis have been collected for the fourth and final time this year. Normal grove care operations continued. These include one herbicide application on July 24 of glyphosate, Kocide was sprayed on August 2 for control of canker, and one application of Intrepid for leaf miner control on August 30.
Within the last year and-a-half, researchers trying to find solution to HLB through production of transgenic plants have used every possible approach that offered some prospect for production of tolerant/resistant citrus plant. As a result, the Core Citrus Transformation Facility (CCTF) has indeed become the platform for testing the effect of different DNA sequences (‘genes’) on transgenic plants that could result in possible alteration of their ability to sustain pathogen attack. Since most of this work in its nature is theoretical, it is not well known how the introduction of certain gene into Citrus will affect production of transgenic plants. Some genes may be easily introduced into model organisms or may cause weak effect on their phenotype. However, very often situation is rather different in Citrus. From one client, CCTF has received a group of orders that all have common DNA sequence combined with other, different DNA sequences. Those orders consisted of six vectors that were supposed to be introduced into three different citrus cultivars in certain combinations. Since the time when these orders were placed and 9/20/2013, altogether 70 co-incubation experiments were performed with more than 38000 explants. Despite all this work, no transgenic plants were produced. From another research group came an order to produce plants with the gene that severely affected the phenotype of transgenic plants. Transgenic shoots were stunted and extremely bushy, making our efforts to graft them impractical. Work on this order stopped after about 30 PCR-positive shoots were produced at which time the agreement was reached with the client who will try to place new order where this gene will be controlled by an inducible promoter. The unintended consequence of the efforts of researchers to find the ‘gene’ that may render Citrus plants tolerant/resistant to HLB by using CCTF as a testing platform is low number of transgenic plants produced in the CCTF. In the last quarter, only 20 plants were produced. They belong to the following orders: pHGJ2 vector-one plant, pHGJ3 vector-seven plants, pHGJ4-three plants, pN5-five plants, pBI121-one plant, pN7-one plant, pW14 one plant, and pMED14 one plant. The second (out of ordinary) reason that contributed to low productivity within this quarter was the massive contamination in one of the common growth rooms where CCTF also keeps germinated plants. This contamination took place in June and July and wiped out 18% of our cultures from that period. Out of three orders for transgenic plants placed in the previous quarter, two were withdrawn. Within this quarter, six orders were placed. Work with two vectors is to proceed only to the early step in shoots production. For additional three vectors, the goal is to establish possible effect of introduced genes on genome activation/transformation success rate. Because of the presence of GFP as a reporter gene in these vectors, the work on these orders may also be completed at the phase of transformation when fully developed shoots are harvested from explants.
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