Recently we have made significant progress in the study of antibiotic resistance in Liberibacter using L. crescens as a model. A former postdoc in the lab, Kin Lai, was able to obtain five spontaneous mutants of L. crescens that were resistant to streptomycin at a frequency of about 1 in 100 million cells (the expected frequency). He was never able to generate spontaneous mutants of L. crescens to oxytetracycline. Kin left the lab and I gave the project to a very talented undergaraduate, Alexa Cohn. Alexa repeated the work of Kin and found the same results. Also like Kin, Alexa was unable to obtain any spontaneous resistant to oxytetracyline even after screening 10e13 cells. But Alexa took the work one step further and discovered something very interesting. She screened for mutants that were simultaneously resistant to streptomycin and oxytetracycline. She obtained two colonies that were resistant to both antimicrobials. Alexa then discovered that all eleven of our streptomycin-resistant mutants were also resistant to both antimicrobials. We are not in the process of getting sufficient DNA form each mutant to sequence each genome to identify the sites of mutation in each strain and then re-create those mutations in wild-type L. crescens using CRISPR technology. This result may have significant management implications. Two antimicrobial products are available to treat citrus for HLB. One contains only oxytetracycline. The other contains oxytetracycline and streptomycin. Our results suggest that treatment with only oxytetracycline is not likely to generate resistance very soon. In contrast, treatment with both antimicrobials may generate resistance to both antimicrobials quickly. We need to work with growers over the next six months to test this notion in the field. This can be done easily once we identify the source of the resistance mutation, which is likely within the rps12 gene. That will allow us to do rapid screening for resistance in the field by qPCR. Our culturing work has also suggested a means by which citrus greening disease might be controlled by a non-antimicrobial means. We have discovered that the preferred carbon source for L. crescens is either alpha-ketoglutarate or citric acid. Citric acid is a common constituent of citrus phloem while alpha-ketoglutarate is not. We are designing experiments to test the hypothesis that if we can nutritionally prevent phloem loading of citric acid, we can then starve Liberibacter in the phloem. We are first testing this nutritional approach in a greenhouse experiment that begins December 12, 2016. This experiment will require two months of nutritional treatment of the saplings followed by another month to extract the phloem, measure organic acids, and interpret the results. We still need a culture of Ca. L. asiaticus (CLas). We have a greatly simplified defined medium for L. crescens that allows the organism to grow much faster than on BM-7, the complex, undefined medium. The necessary changes to this medium are now being made that should encourage CLas growth. Testing of these media will be started within two weeks.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. We repeated the concentration gradient experiments. A series of concentrations of the SAR inducer, including 0, 0.25, 0.5, 0.75, and 1 mM, were used to treat citrus plants by infiltration and soil drench. The infiltrated leaves and soil drenched plants were inoculated with canker bacterial pathogens 24 hours and 7 days later, respectively. Again, 5 plants were used for each treatment; three leaves on each plant were inoculated; 6 inoculations on each leaf were carried out, and a total of 90 inoculations were used for each treatment. Results confirmed that the strength of canker resistance is concentration dependent in the range between 0 to 1 mM. We also confirmed the systemic residual resistance activated by the SAR inducer. The SAR inducer-treated plants were cut back and leaves on the new flushes were tested for resistance to canker. As observed previously, canker disease symptom development was significantly delayed on the leaves on the new flushes. This result indicated that the SAR inducer not only activates resistance in the treated leaf tissues, but also in new flush leaves not treated with SAR inducer. In addition, experiments determining if the systemic residual resistance is conferred by the SAR inducer residue or products induced by the inducer are still ongoing.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. In order to understand how the SAR inducer activates disease resistance, we tested defense gene induction in the treated plants. Citrus leaves were infiltrated with 0. 0.25, 0.5, 1, 5, and 10 mM SAR inducer and the treated leaf tissues were collected at 0, 4, and 24 hours. Expression of a group of defense genes were analyzed by qPCR. These genes include PAL1,NPR1, PR5, CM1, ICS1, CM1, CM2, and PLDg. Results showed that the SAR inducer activated the expression of several defense genes such as NPR1, PR5, and CM1. We also tested if the SAR inducer treatment enhances pathogen induced defense gene expression. Citrus leaves were infiltrated with different concentrations of the SAR inducer. Thirty six hours later, the infiltrated leaves were inoculated with citrus canker bacterial pathogens. The inoculated leaf tissues were collected at 0, 4, 8, and 24 hours later. Expression of the above defense genes were analyzed by qPCR. We found that the bacterial pathogen induced expression of PAL1, NPR1, PR5, CM1, and ICS1 was significantly enhanced by the SAR inducer pretreatment. This results indicate that the SAR inducer can prime citrus plants for resistance to citrus canker. We have also started to test if the SAR inducer can elevate resistance or tolerance to HLB. We are currently testing the treatment conditions.
Between March and June 2016, we followed the proposed research plan by terminating few greenhouse and field experiments, collect and analyze data. I: Effect of drenching application of SL on HLB-infected trees. The soil in potted Valencia trees was drenched with SL at predetermined concentrations. As in the case for foliar applications, tree characteristics were noted and changes recorded on a bi-weekly basis. The second drenching treatment was applied in February as planned and data continued to be collected. The data indicated that drenching application was inefficient in spurring any physical changes as opposed to foliar applications. Despite the double treatment, application of SL via the root system is not efficient. II: Effect of spray application of SL on HLB-infected trees (Repeat experiment). This is a repeat of the experiments involving spraying SL on Valencia potted trees in the greenhouse. Tree characteristics were noted and changes recorded on a bi-weekly basis. Second spray treatment was applied in February and data on flowering, flushing and fruit set recorded biweekly. In parallel to the first year, foliar application of SL oh HLB-affected trees prompted rapid vegetative and reproductive flushes accompanied by the deposition of a wider band of phloem in the roots. This was observed during a routine study of root xylem which walls thicken under HLB. Xylem cell walls in HLB affected trees are considerably thicker then in healthy trees reducing the water conduits. III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. This treatment has been postponed until the arrival of a new scientist to take over the project. He is expected in October, 2016 and the experiments to commence on Nov 2016. IV: Effect of spray application of SL on other varieties of citrus in groves. ‘Midsweet’ was selected as a second variety to be tested for SL effect on tree health. Tree physical characteristics and fruit drop are being monitored throughout. Data on fruit drop was completed after application of spring treatment. The data was similar to those collected the first year. V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic such as organic acids, sugars, amino acids, and few other compounds were applied to trees. So far, diluted sucrose solutions have been effective in enhancing new flush in trees with advanced stages of HLB. Sucrose has been applied to trees in a monthly basis and some recovery has been observed. Treatments continue but improvement has slowed down.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). The following work has been carried out in this quarter: (1) Inoculated the promising candidate transgenic plants with CTV carrying the flower-promoting gene FT3. (2) Propagated the transgenic line HAM 13-3, DUN 57-25, DUN205-25c, and DUN 207-8. (3) Infected progenies of transgenic plants with Las-carrying psyllids. We have generated more transgenic plants expressing various defense genes. These transgenic plants are growing in greenhouse and will be tested once they are ready.
During this reporting period July, August, and September, 2016), Dr. McNellis continued to work with USDA APHIS to obtain permitting to transfer a set of ‘Duncan’ grapefruit plants expressing the FLT-antiNodT fusion protein from Penn State University to Fort Detrick in Frederick, Maryland. These plants are to be tested for resistance to HLB using a psyllid-vectored inoculation system in a secure greenhouse. We anticipate approval during the next reporting period. In addition, Dr. McNellis’ team at Penn State continued to evaluate the solubility and stability of the FLT-antiNodT fusion protein in citrus extracts and presence of the FLT-antiNodT fusion protein in various plant tissues by protein gel immunoblotting. The FLT-antiNodT fusion protein appears to be produced and present in all tissues examined to date, although these tests are ongoing and will continue into the next reporting period. Dr. McNellis presented a poster describing the results of the project to date at the annual conference of the American Phytopathological Society in Tampa, FL, July 30 – August 4, 2016. A poster viewer at the conference had some suggestions as to how to determine whether the FLT-antiNodT fusion protein indeed binds to its target in vivo, and Dr. McNellis has developed an experimental plan for doing this, which will be initiated during the next reporting period.
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. 1. To test for he unlikely but increasing possibility that HLB is transmitted by extracellular vectors, we isolated DNA from HLB leaves and inject these into 2 year old Valencia trees. The trees are being kept in a greenhouse and are under observation. As of June 2016, trees were growing normally. Samples of nectar, honey, pollen, albedo, flavedo and flowers collected in spring time were were analyzed. Albedo, flavedo and flower buds all tested HLB+, whereas pollen, seeds, nectar and honey were all HLB-. 2. Experiments for objective 2 are well under way. Two trees (one healthy and one HLB+) were root grafted in three different locations and placed in special pots large enough to accommodate the 2 trees. The trees have been placed in a greenhouse and continue currently under observation. PCR analyses were conducted in May 2016 and one of the 5 originally healthy trees tested positive, although clear visible symptoms were not evident. 3. Grafted trees with HLB material are being monitored weekly using Narrow-band imaging under polarized illumination. Although we continue to have issues with the background, we have established a standard curve and a correlation relationship between starch levels, PCR values, and polarized light readings. 4. Trees have been grafted for a substantial amount of time and some started showing HLB symptoms. However, given that analysis of this objective destroys the trees, more time is needed to be certain that HLB has taken root. PCR analyses was performed in May 2016 and no tree tested positive despite the now clear symptoms of HLB. In general, we continue to monitor all experiments realizing that CLas titer drops during the summer. All trees will be tested again in September and proper action taken depending on the results.
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. 1. To test for he unlikely, but increasing, possibility that HLB is transmitted by extracellular vectors, we isolated DNA from HLB leaves and inject these into 2 year old Valencia trees. The trees are being kept in a greenhouse and are under observation. As of June 2016, trees were growing normally. Trees tested in September 11, one tree testing HLB+, though a high PCR value. Samples of nectar, honey, pollen, albedo, flavedo and flowers collected in spring time had been analyzed previously. Albedo, flavedo and flower buds all tested HLB+, whereas pollen, seeds, nectar and honey were all HLB-. 2. Experiments for objective 2 are well under way. Two trees (one healthy and one HLB+) were root grafted in three different locations and placed in special pots large enough to accommodate the 2 trees (5 pairs). The trees were placed in a greenhouse and kept under observation. PCR analyses were conducted once more in September 2016. At this time, 3 out of the 5 pairs of the initially healthy trees tested positive, although clear visible symptoms were not evident in all cases. 3. Grafted trees with HLB material are being monitored weekly using Narrow-band imaging under polarized illumination. Although we continue to have issues with the background, we have established a standard curve and a correlation relationship between starch levels, PCR values, and polarized light readings. 4. Trees have been grafted for a substantial amount of time and some are showing HLB symptoms. However, given that analysis of this objective destroys the trees, only trees with clear symptoms are tested. PCR analyses was conducted in one tree using using all leaves in 2 complete orthostichies. In this particular tree, there was no correlation between orthostichy and titer, although there PCR values were significantly lower in the laves above the infection point.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol may need to be optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. A construct targeting a site overlapping the two conserved leucines has been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. A replacement recessive bs5 allele will be added, and this construct will be prioritized for transformation into Carrizo citrange for proof of concept. Resulting plants with biallelic mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol may need to be optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. A construct targeting a site overlapping the two conserved leucines has been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. A replacement recessive bs5 allele will be added, and this construct will be prioritized for transformation into Carrizo citrange for proof of concept. Resulting plants with biallelic mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
During the reporting time period, we were in the process of hiring a scientist to work on project. Meanwhile, we have made following progresses for the proposed objectives: We analyzed and identified the gene sequences required for the projects and designed all proposed gene constructs. Most of these gene cassettes are currently under construction, with some being finished soon. These constructs will be used in citrus transformation to test their effects in promoting mature citrus transformation. We have also been evaluating the effects of a hormone related gene in improving micro-grafting efficiency using tobacco as a quick testing model plant. Our results demonstrate that the expression of that particular hormone related gene in rootstock plants can improve scion grafting success rate, reduce rootstock’s lateral bud release, and enhance rootstock’s root initiation. We will soon ship this gene to Dr. Janice Zale of the Mature Citrus Facility at the University of Florida for them to test its effects on citrus.
Citrus trees transformed with a chimera AMP and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Propagated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Twelve months after graft inoculation, Las titer was examined and compared in old leaves (most with HLB symptom), young expanded leaves (with or without HLB symptom) and fibrous roots of transgenic and control plants. Our results showed again that transgenic citrus expressing Mthionin has lower Las titer (200-1800X lower) compared to control and transgenic plant expressing chimera. These data suggest transgenic plants expressing thionin are promising for HLB resistance ( published in Frontiers in Plant Biology). Antibody against thionin has been produced for investigating the correlation of thionin expression and HLB resistance. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic carrizo plants carrying second generation AMPs were obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. Furthermore, the third generation chimeral peptides were designed based on citrus thionins, the vector construction were finished and citrus transformation are underway. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA was isolated and Las titer was tested. Our preliminary results showed that transgenic trees expressing NbFLS2 can reduced Las titer. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las tilter will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
Additional UF trees have been planted in the last quarter. Data collection continues across numerous experiments. A number of publications from UF and USDA have included data from these plantings. A 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 six years and new trees are being added every few months. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015. The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Ramadugu et al, Plant Disease, 2016), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree (Miles et al., 2016). All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. A new UF-Gmitter led association mapping study has underway using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
The overall objective of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using Liberibacter crescens strain BT-1. We have accomplished that goal and used it to screen potential phage, tailocins and microbial compounds for activity against the model bacterium L. crescens BT-1. Since Liberibacter is a member of the Rhizobiaceae we have recently taken the approach that phylogenetically related microorganisms can share common surface components, such as phage receptor sites. Analysis of major outer proteins of Candidatus Liberibacter spp. , that are known to be expressed in planta and in the vector, identified several proteins that could potentially act as adsorption sites for phage and/or tailocins. Bioinformatics and structural analyses indicated that there is ~40% identity and ~61% homology of OmpA and TolC outer membrane proteins between Rhizobium spp., Agrobacterium spp. and Liberibacter spp. Therefore, our strategies has been to search for naturally occurring phages active against Rhizobium spp. and/or Agrobacterium spp. that may also show activity against Liberibacter spp. We have isolated a bank of three Agrobacterium spp. and 15 Rhizobium spp. phages. The Agrobacterium phages exhibit differential activity among Agrobacterium spp. hosts, as do the 15 Rhizobium phages, which indicates a diversity of receptor sites. One particular Rhizobium phage, R2phi3LR, forms plaques on both Agrobacterium spp. and Rhizobium spp. hosts, which is indicative of common receptor(s). As expected, the efficiency of plating for phage R2phi3LR was reduced 1000X when Rhizobium propagated phage was titered on Agrobacterium and vice versa. All purified phages are being increased to conduct plant studies that will evaluate the potential activity of the phages against Candidatus Liberibacter spp. .
This project is a continuation of CRDF 447 to evaluate effects of Metalized Reflective Mulch (MRM) and insecticides to protect newly planted trees from the Asian Citrus Psyllid (ACP). While the previous project demonstrated that trees planted over the MRM treatment exhibited less ACP populations, less HLB symptoms and greater growth, it did not include crop yield and fruit quality differences which is the focus of this project. Specifically the objectives of this project are: 1) To determine if grapefruit trees planted into beds covered with Metalized Reflective Mulch (MRM) come into viable crop production at a younger age than in conventional plantings based on yield differences. 2) To continue to document tree growth differences between the three treatments: bare ground (convention grower standard), compost applications, and MRM and to monitor (weekly) insect pest populations to determine the insect control benefits of the MRM. 3) To determine the HLB incidence with PCR analysis for each treatment to verify the effectiveness of MRM to bring young trees into production without HLB symptoms. 4) To determine tree condition by improvements of tree health attributable to MRM will be made by visual assessments and Normalized Difference Vegetation Index (NDVI) determined by aerial (UAV) imagery of the canopy to determine the tree condition for each treatment. 5) To record and document production costs and economic returns for each treatment. To accomplish these goals we have continued to make growth measurements for the following parameters: tree caliper, tree height, canopy diameter and canopy volume. In all these four growth measurement the MRM treatments were significantly greater than both the bare ground and compost treatments and in almost all case the increase of the MRM treatment was double that attributed to the compost treatment when bare ground was the control treatment. For example the increase in canopy volume for MRM was 137% greater than bare ground while compost was 72% greater. The Volumetric Water Content (VWC) for the three treatments was sampled monthly and the MRM consistently remained the highest for the three treatments. Tree health was evaluated visually as tree condition and the MRM treatment trees has the lowest percentage of weak trees (1.6%), Compost (3.8%) and Bare Ground (18.8%). Similar trends were observed for symptomatic conditions attributable to HLB. The MRM treatment continued to offer lower ACP counts for all life stages (eggs, nymphs and adults) based on weekly scouting. Two other insect pest populations were also observed to be lower in the MRM treatment were Diaprepes Root Weevil and Orange Dog larvae. A detailed spreadsheet has been created to track all the caretaking expenses for the trial block including all spray foliar spray applications, herbicide treatments, fertilizer applications, soil drenches as well as the associated materials. These caretaking events are entered as they are accomplished and are combined to yield the total production cost for each treatment. Pertaining to yield component of the trial we have met with Dr. Alan Wright and Mr. Jerry Britt (IRREC) to coordinate the upcoming harvest event to determine the average fruit yield and fruit size for each treatment by means of a portable scale and fruit sizer. The average fruit drop per tree was assessed for each treatment and yielded the following data: Bare Ground 3.6, Compost (UPD) 3.2 and MRM 2.3. Currently the overall tree condition trial is deemed to be excellent with very minor damage associated with Hurricane Matthew.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
