The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). Our previous study indicate that Las contains a functional SA hydroxylase that degrades SA and its derivatives. SA and its derivatives play important roles in plant defenses. Las employs SA hydroxylase to suppress plant defenses. Our central hypothesis is that we can improve HLB management by counteracting SA hydroxylase. We will focus on counteracting SA hydroxylase using inhibitors based on structure based design. This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied once onto selected trees by foliar spray in November, 2015 during fall flush. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse.
The goal of the proposed study is to characterize the effect of using endophytic microbes in controlling HLB. Our hypothesis is the outcome of the interaction among Las, psyllid and citrus is affected by the citrus phytobiome. In order to achieve the goal of this study, the following objectives will be conducted: Objective 1. To characterize the phytobiomes and endophytic microbes from HLB survivor trees and HLB diseased trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. Rhizosphere, rhizoplane and endosphere DNA and RNA preparation was done using powersoil RNA extraction kit. The DNA and RNA samples were sequenced. Around 10 Gb clean reads data was generated per metagenome sample. Analysis of the metagenomic data is ongoing. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. We have begun to test different grafting methods of roots. We are also in the process of isolating endophytic microbes associated with survivor trees.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We will mainly test the antagonistic effect of Bacillus, Paenibacillus, Streptomyces and Pseudomonas strains Agrobacterium tumefaciens, Sinorhizobium meliloti, and L. cresens; Control HLB using antibacterial-producing bacteria. For the field test, we will investigate how antibacterial-producing bacteria affect HLB disease severity, Las titres, and citrus yield, survival of the antibacterial-producing bacteria in the rhizosphere and expression of the antibacterial biosynthesis genes in vivo. We have isolated Streptomyces spp. Bacillus spp. Paenibacillus spp., and Pseudomonas spp. from Florida groves. Multiple isolates showed antimicrobial production activity. We tested 27 antibacterial compound producing bacteria. These strains had been recovered, purified and confirmed by 16S rDNA sequencing. The antagonistic activity against Agrobacterium, Sionrhizobium meliloti and Xanthomonas citri pv. citri was determined. 5 strains, belonging to Paenibacillus, Burkholderia, Paenibacillus, Streptomyces and Streptomyces showed good antagonistic activity. Three bacteria showing high antimicrobial activities have been sequenced to help us understand the mechanism. Currently, the genome sequencing was finished and we are analyzing the results. Four bacterial strains: two Burkholderia, one Pseudomonas geniculata, one Rhodococcus strains have been tested for their activity in and all showed induced plant defenses and against infection by Xanthomonas citri. To further study the antimicrobial producing bacteria, tow Burkholderia strains have been labeled with GFP tag. Seven other strains are being labeled with GFP or RFP tag. We also investigated the antibiotic genes in nine antimicrobial producing bacteria that we isolated previously. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi). In a separate experiment, two Burkholderia strains were tested and up to 10E5 cfu/g soil was recovered at five days post inoculation. For the field trial, we have selected the grove and conduct survey on HLB disease severity. We compared different delivery methods to improve the efficacy of beneficial bacteria. A root drench delivery method has been established. Pseudomonas geniculata strain 95 was applied to trees in Block 9, CREC on Oct 22, 2015. And the bacteria are applied every two months. Three concentrations: 10E6, 10E7, and 10E8 CFU/ml were used and 1 gallon bacterial culture was applied per tree using soil drench method. We are investigating HLB disease index, bacterial survival rate and Las titers.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgeniclines showed delayed canker symptom development. Further detailed analysis of transgenic plants is being carried out.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (Oct 2015 to Dec 2015), we continue to evaluate 1) the effect of Pen and SD on control of HLB disease by gravity bag infusion in the field; 2) the efficiency of effective chemical compounds (Pen, SDX, Pcy and Carv) against HLB disease by gravity bag infusion; 3) the effectiveness of a combination of chemotherapy, thermotherapy and nutrient therapy against HLB in the field trials. The chemical compounds (Pen and EBI-602) and additional nutrients were applied to the heat-treated citrus for two times by foliage spray, using our optimized nano-delivery system. The variance analysis results indicated that there were significant differences in the chemical treatments, the heat treatments and their interactions. Pen was the more effective to control Las bacterium than EBI-602. In this quarter, ten chemicals were prepared in two different concentrations of the nano formulations (0.1% and 1.0%) in the greenhouse test. The preliminary results showed that the concentrations of nano formulations had no different effects on the chemicals for control of Las bacterium. PEN and ZS were the most effective to eliminate the Las bacterium. The effects of the integrated approach on the citrus fruits will be reported in next quarter.
The use of antimicrobials is one of the few effective treatments against HLB in citrus trees. However, penetration of substances into trees is hindered by the presence of protective layers such as the thick cuticle on leaves, and cork on stems. To overcome the obstacles imposed by the cuticle to increase penetration of externally supplied substances, we have successfully tested laser light. Laser light technology involves the use of low level light energy to disperse the cuticle creating microscopic and superficial indentations of approximately 250 m. In doing so, infiltration of substances into the leaf is greatly enhanced. Once inside the leaf tissue, substances can follow the natural transport pathway through the apoplast, absorbed by phloem cells, and transported throughout the tree. Specific goals are 1. To build and test a more flexible and elaborate laser machine that will allow for more decisive experiments in the greenhouse; 2. Test for the effectiveness of several antimicrobials; 3. Carry out initial field experiments with young trees. The laser machine was delivered and tested under laboratory conditions. A series of initial experiments were conducted in efforts to fine tune the machine in terms of energy, distance, speed of laser and striking angle. Additional experiments have been conducted comparing the effectiveness of laser in enhancing penetration of oxytetracycline compared to untreated leaves. We have also tested the effect of waxing the lasered area to prevent dessication. In some key experiments we have tested the changes in titer numbers of samples treated with antimicrobial in the presence and absence of laser. At the moment, the data collected indicate the superior penetration and effect of antimicrobials when leaves are subjected to a laser treatment. Additional experiments to quantify the penetration improvement and movement of oxytetracycline down the phloem are being conducted.
This is a 3 year study to provide a side-by-side comparison of 5 soil-applied commercially available products as well as an organic mulch as recommended by growers. The impact of treatments on tree health, foliar nutrition, disease rating, HLB status, root density, yield and fruit quality will be evaluated. Trials will be located in 3 Florida sites: Central Ridge, East Coast and Southwest and will incorporate well-managed trees with HLB but that are as healthy as possible. Each trial will be large enough to be statistically significant with treatments within label guidelines to avoid need for crop destruct or permitting. Objectives of the Project: Test 5 soil-applied products, with mulch subplots, plus an untreated control (6 treatment plots) on health and HLB status of orange trees over 3 years. The 6 treatment plots of 20 trees each will be replicated 4 times plus mulch subplots at the 3 sites will include: 1. BioFlourish (Triangle Chemical).3 applications per year; 2. Ecofriendly Citrus Soil Amendment. 8 x per year; 3. Serenade Soil (Bayer CropScience) 3 x per year; 4. Aliette (Bayer CropScience) 3 x per year 5. Quantum product line 12 x per year; 6. Untreated control (3 mulched trees only 1/year) Our responsibility is to analyze microbe products, analyze root mass, Las in the root, survival or colonization of microbes in the roots, and indicator strains of microbial diversity in the soil. Totally 5 products, including Bioflourish, Ecofridendly citrus soil amendment, serenade soil, aliette and quantum product were involved. The bacteria ingredients in the product Bioflourish and Ecofridendly citrus soil amendment were determined. There were several Lactobacillus species in Bioflourish, and Bacillus subtitis was detected in Ecofridendly citrus soil amendment. The bacteria in serenade soil and quantum product were mentioned by the producer and thus not tested in lab. The field trails were carried out at three sites around Florida (south west, central ridge and east coast) as described by the proposal. The root density was determined in December 2014. Roots were extracted from 500 cc aliquant of each soil sample over a 2 mm mesh sieve by hand. The dry weight of roots was measured. There were totally 504 samples (3 sites*6 treatments*4replicates*7 trees). The average root mass was 1.14 g/500 cc soil. The root mass did not show any significant difference between treatments, neither for each site nor pulled together (ANOVA, P>0.05). When mulch treatments were taken into account, the results suggested mulched treated trees tended to have higher root mass, but not significant (ANOVA, P>0.05).
This is a 3 year study to test 5 soil applied treatments to prevent HLB infection in newly planted citrus trees and also the test the ability of treatments to maintain tree health should they become infected. The materials are to be Product A – Numbered Compound. Product B – Aliette Product C – Serenade Soil as well as 1 combination of A+B. The impact of treatments on new tree health, foliar nutrition, disease rating, HLB status, root density, subsequent yield and fruit quality will be evaluated. The trial will be located at a single Florida site and will incorporate well-managed trees from a commercial nursery in a new planting. This trial will be large enough to be statistically significant with treatments within label guidelines for products B and C which are registered for citrus. Our main responsibility is to examine annual root density and Las population in the root in year 2 and year 3. Product C contained Bacillus subtilis as active ingredient, so the root colonization and survival rate in soil were tested under greenhouse conditions. 15 ml of product C was applied to Valencia seedlings. The roots and rhizosphere soil were harvested at 1dpi (days post inoculation), 7 dpi and 15 dpi for root colonization and survival analysis. The analysis for other products is ongoing. Product A was not available, product B did not contain bacteria. The samples from project 934.1C were just received. The root mass and bacterial community of each treatment will be determined
The use of antimicrobials is one of the few effective treatments against HLB in citrus trees. However, penetration of substances into trees is hindered by the presence of protective layers such as the thick cuticle on leaves, and cork on stems. To overcome the obstacles imposed by the cuticle to increase penetration of externally supplied substances, we have successfully tested laser light. Laser light technology involves the use of low level light energy to disperse the cuticle creating microscopic and superficial indentations of approximately 250 m. In doing so, infiltration of substances into the leaf is greatly enhanced. Once inside the leaf tissue, substances can follow the natural transport pathway through the apoplast, absorbed by phloem cells, and transported throughout the tree. Specific goals are 1. To build and test a more flexible and elaborate laser machine that will allow for more decisive experiments in the greenhouse; 2. Test for the effectiveness of several antimicrobials; 3. Carry out initial field experiments with young trees. The laser machine was ordered, and delivered in early September. The machine was made compatible with UF system and connected to a new computer especially purchased for this purposes. The basic machine is flexible in terms of mobility and capable of adjustments in all three dimensions X, Y and Z. This capability makes it user friendly in the lab as well as in the greenhouse. At the end of the month, we fine tunned the machine in terms of laser energy, laser speed and focal distance. At the end of the reporting period, we had become familiarized with the machine and were ready to do some greenhouse testing.
September 2015 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. Experiments to confirm initial relative humidity findings continue. A new set of twigs was inoculated and incubation started but it is unlikely to see structures for at least 4-6 weeks. An employee was hired to assist with this project. A site has tentatively been found to conduct field experiments of inoculum potential and some preliminary work is underway. Assays to determine the survival of P. citricarpa pycnidiospores following exposure to temperatures ranging from 5 to 50 C, for periods of 1 to 48h were completed. We have determined that spore germination increases with increasing temperature to a maximum at 25 C and declines at 30 C and above. Over time, incubation periods of up to 3h do not have an impact on the survival of the pycnidiospores at temperatures of 15-30 C. Across all time and temperature treatments, exposure of spores to drying and subsequent low relative humidity was significant, essentially eliminating spore viability. These results are providing valuable information for modeling the potential for spore survival in and on equipment associated with grove operations. Additional experiments to assess the environmental conditions associated with optimal pycnidiospore production have been established and are ongoing. Preliminary results suggest no, or only a modest influence of light on spore production in culture and experiments to test the influence of temperature and relative humidity are ongoing.
September 2015 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. In the large field trial, there was ten times more G. mangiferae than G. citricarpa in the leaf litter. In 2014, there was no pattern in the number of leaves with Guignardia structures over time in any treatment. The treatment with the greatest number of leaves with structures was urea over all. In 2015, the pattern of structure formation was consistent across treatments and over all the treatment with the fewest structures was urea. The DNA analysis of the 2015 leaf litter is continuing. The extractions are complete but the PCR is not complete The bagasse field trials confirmed the laboratory experiments that bagasse increased the leaf decomposition rate compared to nothing or urea. Greater soil moisture also accelerate leaf decomposition. The manuscript preparation is still not complete. Collection of leaf samples from the grove in Immokalee has continued biweekly. Each batch of samples contained 40 samples of 25 leaves collected below 40 trees. Leaves were examined under microscope to check for fructification of Phyllosticta spp. Leaf portions without fructification were discarded and the remainder were immersed in 0.02% tween20 to collect conidia and ascospores. Conidia and ascospores produced in leaf litter were quantified, weather data were collected from FAWN. Data collection continues and we are trouble shooting the qPCR to make sure that it is working properly before samples are processed. In Australia, work continues on the mating and production of ascospores for Phyllostictam citricarpa in culture. None were formed on the leaf discs but pseudothecia were formed using another technique. Confirmation of the results is under way. Inoculations of fruit are underway in the field. They are repeating the fungicide work to confirm previous results. They are also continuing to sample leaf litter in two groves in Queensland mandarin growing region.
September 2015 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: Manuscript preparation continues for the DMI fungicides. Media were selected for experiments with SDHIs and the assays with Endura are nearly complete. Other SDHIs are underway. Experiments with Cannonball and Vanguard are continuing. The mycelial tests are complete and spore germination assays are still on-going. Objective 2: Fungicide trials were initiated in 2015. Since July 2 applications were made for the fungicide trial. The disease is in its latent phase. Objective 3: We are waiting for the disease to start expressing for data collection Objective 4: It was found that Eugenol volatiles alone, whether 50 L/L or 100 L/L, did not inhibit mycelial growth of any of the three Guignardia citricarpa isolates tested, but it did decrease the number of conidia formed on colonies for all three isolates and in a dose-dependent manner (P < 0.05). After 14 day incubation, exposure to 50 L/L of eugenol volatile decreased the number of conidia from isolates CNGC, PPST, or GC1 by 79%, 54%, or 79%, respectively, and exposure to 100 L/L of eugenol volatiles decreased the number of conidia of the above three isolates by 90%, 74%, or 78%, respectively, compared to the control.
The three growers have applied the 1/4 rates of Citrus Fix (2, 4-D), MaxCel (cytokinin) and now also ProGibb every 45 days on approximately 1 acre each of Hamlin and Valencia orange trees. Treated and Control trees are categorized by three levels of tree health and are being monitored for fruit drop, leaf drop, flushing, yield, etc. One other location is receiving similar treatments in a replicated trial. We are monitoring leaf drop to see if these frequent applications may affect leaf drop as well as fruit drop. If they do, it would more than compensate for keeping some additional fruit on the tree longer than normal for HLB affected trees.
The last application of low concentrations (1/4 rate) of Citrus Fix (2, 4-D) and MaxCel (Cytokinin) with or without ProGibb (GA) every 45 days to Hamlin and Valencia tree canopies in central Florida for the 2015-16 growing season are completed. One of the two sites will end this year as the grower has chosen to discontinue production inputs at this site. A second test of applications GA to roots of nursery trees infected with HLB was started and two 4 week cycles will be completed about November 1st. The trees had been infected in a hot psyllid greenhouse. GA stimulated root growth on healthy trees, now we can determine if it can stimulate root growth on infected plants.
All the data from flowering branches was summarized for 4 Hamlin, 4 Valencia, two Murcott and two Sunburst blocks in various locations from Frostproof to Auburndale, FL. The best way to use this data in the Citrus Flowering Monitor System is now being assessed. The on-line ‘Citrus Flowering Monitor System has been upgrades and now covers new FAWN sites and provided accumulated induction hours and dates of bud break and full bloom. The fist estimates of vegetative flush will be added in the next two months along with 5-10 % open flower estimates. This will be in time to test it this coming spring.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
