Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1160 - Pathology | 50% |
| 402 - Engineering Systems and Equipment | 999 - Citrus, general/other | 1160 - Pathology | 50% |
This four-year multi-state multi-disciplinary research and extension project aims to address the urgent need of citrus growers in Florida to an immediate treatment for HLB-affected citrus trees by utilizing steam for rapid thermotherapy. The overall goal of this project is to provide an immediate scalable technique using steam as well as synthesize scientific explanations on how the technique works in mitigating HLB and prolonging the production life of infected citrus trees. The central hypothesis of the project is that the HLB-affected trees can be treated for inoculum reduction using a rapid steam-based thermotherapy technique in a manner that is biologically and economically feasible with minimal environmental impact. This project covers both the practical and theoretical aspects of the thermotheraphy through an interdiscplinary approach involving researchers with expertise in plant pathology, plant physiology, engineering, nematology, economics, and plant biology. The project objectives include enhancement of the steaming system and operations used for the treatment, investigation of the effects of steam-based treatments on the survivability of CLas and the expression of HLB symptoms, determination of treatment efficacy and effectiveness, development of a comprehensive steaming system which includes treatment of the roots, studying the effects of heat treatment on yield and fruit quality, and conducting comprehensive economic analyses of the system. The enhanced steaming system will be used to treat citrus trees on a quarterly basis from different sites in collaboration with local citrus growers. An strong extension program is planned to disseminate the outcome of this research.
Objective 1:An existing heat treatment machine will be enhanced to provide rapid, precisely controlled, and uniform supplementary heat treatment into the canopy, roots, and surrounding soil of HLB-affected trees. Associated tasks include: (a) construction of an enhanced steaming system, (b) development of a mathematical model to predict both the heat transfer through the tree and the soil as the steam is applied and the temperature along the depth and direction of canopy and soil, (c) design and construction of a feedback control system to efficiently control the heat thermotherapy process, (d) development of a new mechanism for heat treating the root system, and (e) development of a post-treatment mineral oil application to improve the effect of the steam thermotherapy (objective 6).Objective 2:The steaming system from objective 1 will be used to conduct a comprehensive field trial in 'Valencia' sweet orange. The trial will be designed as a split-plot design with timing of treatment application being the main-plot. Main-plots will be steam applications done at quarterly intervals to determine if there is an optimal time of year for CLas reduction in the canopy. The main-plots will be laid out as either a completely randomized design (CRD) or a randomized complete block design (RCBD) depending on the aspects of the site selected for the experiment. The main plot will consist of a minimum of 50 trees, subdivided into 10 tree sub-plots. There will be five replications of the main plots. For the first year of study, the sub-plot treatments will include five levels of heat treatment: 1) no heat treatment (control); 2) 58°C for 15 s; 3) 58°C for 60 s; 4) 58°C for 90 s; 5) 58°C for 120 s; and 6) 60°C for 30 seconds. These heat and time combinations have been selected to create two extreme set-ups including one combination in which steam is applied but no defoliation occurs while the other combination will cause total defoliation based on prior experiments. Before the steam treatments in each main-plot, an HLB symptom assessment (scale 1= Vigorous, asymptomatic, 2= Slight decline, symptomatic, 3= Moderate decline, symptomatic, 4= Severe decline, symptomatic, 5= Nonviable, will not recover) and sampling for CLas will be done.Objective 3:The viability of CLas after heat treatments will be used to assess the effectiveness of the treatment to reduce or eliminate the bacteria in the tree. Two different approaches will be used to assess bacterial viability. One will be based on the propidium monoazide (PMA) real-time PCR assay adapted for CLas detection.The second approach to assessing CLas viability will be based on a comparison of specific mRNA populations in the bacteria either with total genomic DNA (CLas) or CLas 23 rRNA levels. The rationale is that mRNAs in bacteria (and other organisms) show much higher turnover rates compared to genomic DNA or 23S rRNA. Hence, fluctuations in mRNA population size can provide a sensitive means to assess the overall viability of bacteria soon after the heat treatment. The expectation is that the exposure to high temperatures will result in drastic changes in the pattern of RNA abundance within minutes to hours after treatment.Objective 4:Sub-objectives related Objective 4 are to i) characterize the effects of the treatment on soilborne pests and diseases and ii) determine whether additional IPM practices improve the thermotherapy outcome. In addition to the field experiment described in Objective 2, we will select groves based on the infestation status for three key pests of the citrus root system: the root weevil Diaprepes abbreviatus the phytoparasitic, nematode Tylenchulus semipenetrans, and the oomycete Phytophthora nicotianae. Tree responses to thermotherapy in groves infested and not infested by each pest will be compared. Additionally, in some infested groves, thermotherapy will be applied to trees that are treated or not treated with chemical and biological pesticides capable of reducing pest population densities.Objective 5:Sustained productivity of citrus trees after heat treatment requires that the metabolic functions of the tree be maintained and that CLas populations be reduced to minimize symptom development and tree decline; our work indicates both are possible. Two key underlying factors for the long-term success of thermal treatment will be established. First, mathematical functions that describe the amount of time that a leaf or twig can be exposed to steam at a given temperature without dying will be derived and verified for CLas inactivation in planta, and a rapid enzymatic method developed for assessing leaf viability. Understanding the rate of heat transfer between steam and trees is critical to establish a dependable system. In the proposed system, as steam is injected into the canopy, convective streams of steam and air mixtures are produced; optimization of the system requires characterization of heat transfer to individual tree components. Secondly, to understand the thermal death kinetics of CLas in citrus branches, D-values and the z-value will be established. When microorganisms are heated at a constant temperature, the decrease of viable organisms follows a first order reaction, commonly defined by D-values (the time to inactivate 90% (1-log) of microorganisms). The temperature dependence of D-values are represented by z-values (the temperature change needed to change D-values by a factor of 10). D- and z- values allow for the determination of equivalent rates of microbial kill under varying time and temperature treatment combinations.Objective 6:In this objective, the effect of post-treatment with three HMO aerosols to improve effectiveness of thermotherapy on HLB-affected canopies will be evaluated. The HMO selection, application volume, and concentration (
Target Audience
Citrus growers, Crop consultants, Agricultural machinery industry. Growers are seeking novel ways to effectively mitigate the negative production effects from citrus greening (i.e. HLB). Early trials using thermotherapy techniques showed promise in suppressing the bacterial effects of HLB and enhancing renewed tree growth and fruit production. This project centered around a large-scale trial where high-temperature steam "cooked" individual trees for varying durations to see if this thermotherapy technique could achieve economic feasibility. If positive trial results were observed, equipment manufacturers would be encouraged to design and build cost-effective treatment systems to apply the thermotherapy treatment.
Changes / Problems
Hurricane Irma affected our experiments and measurements. We asked for a no-cost extension.
Training & Professional Development
Graduate students and postdocs have had training opportunities and developed the mobile thermotherapy system, the sensing system and the thermodynamic model. They presented their work in international conferences. Several graduate and undergraduate students, and postdocs worked on this project and evaluated the thermal treatments, and participated in the preparation of oral and poster presentations. For example, one graduate student has had training opportunities and has worked on RNA methods and interpretation as well as professional development presenting results at regional and international meetings. Two postdocs have had one on one training opportunities and developed experimental design, data analysis, and presentation skills while studying conditions that induce movement of Candidatus Liberibacter asiaticus between the roots and canopy to better guide application timing. Furthermore, we demonstrated the developed mobile thermotherapy system, presented our results, demonstrated EPN application techniques (among other activities) to growers and stakeholders.
Dissemination Streams
As part of the extension effort to inform citrus industry members, master gardeners, and homeowners about research outcomes and HLB management we presented material from this project at several conferences (see journal and conference publications), trade shows, workshops, and field days; trade journal articles were written explaining the results too. We also handed out HLB identification and management informational documents at many events. As an example: Extension Publications Ampatzidis, Y. 2018. Agricultural engineering technologies to improve citrus production, Citrus Show, Fort Pierce, Florida, Jan-24, 2018. Ampatzidis Y. 2018. Smart Machines, UAV and Precision Farming Technologies, 2018 Citrus Innovation and Technology Show. Arcadia, Florida, April-5, 2018. Ampatzidis Y. 2018. Agricultural and Biological Engineering Technology Applied to Citrus Crops, 2018. Citrus Health Forum, NFREC, Quincy Fl., April 19, 2018. Ampatzidis Y. 2018. New Technologies in Agriculture for Specialty Crops, 2018. Citrus Engineering Conference, Panel Session, Lake Alfred, FL, June 6, 2018. Ampatzidis Y. 2018. Smart and Precision Agriculture, 2018. LaBelle Rotary Club, LaBelle Fl, September 17, 2018. Ampatzidis Y. 2017. UF Citrus Field day, Immokalee, FL, USA, Nov-2, 2017. Dewdney, M., Johnson, E., Thapa, N., and Danyluk, M. 2018. Thermotherapy's effects on fruit drop, yield and quality. Citrus Industry 99(12): 12-15. Ehsani, R., Dewdney, M.M., and Johnson, E.G. 2016. Controlling HLB with thermotherapy: What we have learned so far? Citrus Industry 97(9): 26-28. Ehsani, R. and Pertiwi, C. 2015. Tenting and supplementary heat for thermotherapy of HLB trees. Citrus Industry 96(8): 8, 10, 12. Thapa, N., Johnson, E.G., and Dewdney, M.M. 2019. Effect of thermotherapy on fruit yield. Citrus Expo. Fort Myers, Florida. Thapa, N., Johnson, E.G., and Dewdney, M.M. 2019. HLB and steam thermotherapy. Citrus Expo. Fort Myers, Florida. Johnson, E. G. 2018. What's going on below ground? Citrus Growers' workshop. Sebring, FL. September 25, 2018 Johnson, E. G. 2018. Effect of HLB on citrus root density. Citrus nutrient management for trees affected by HLB. Immokalee, FL. February 2, 2018. Johnson, E. G. 2017. Root Health of HLB-affected citrus. Citrus Show. Ft. Pierce, FL. January 26. Johnson, E. G. 2017 Rootstocks, root lifespan, and yield: What do they tell us about managing root health with HLB. Citrus Production School. Arcadia, FL. February 28. Johnson, E. G. 2017. Citrus Root Health and HLB Management. Citrus Institute. April 4. Johnson, E. G. 2017. Citrus Root Health Management. Citrus Expo. August 17. Johnson, E. G. 2016. Root Health in Florida Citrus Trees. Ridge Citrus School. October 25. Johnson, E. G. 2016. Video of root responses to HLB using minirhizotrons. UF/IFAS/CREC extension booths. First displayed at the Citrus Expo August. Ehsani, R., Trotochaud, J. and Souri, S. 2016. Heat therapies. Citrus Expo. Fort Myers, Florida. Ehsani, R. 2016. Heat therapy overview. The Citrus Show. Fort Pierce, Florida. (revised) Ehsani, R. 2016. Comparison of heat therapy methods. The Citrus Show. Fort Pierce, Florida. (revised) Ehsani, R. 2016. On-going heat therapy work. The Citrus Show. Fort Pierce, Florida. (revised) Ehsani, R. 2015. Heat therapy overview. Citrus Expo. Fort Myers, Florida. Ehsani, R. 2015. Comparison of heat therapy methods. Citrus Expo. Fort Myers, Florida. Ehsani, R. 2015. On-going heat therapy work. Citrus Expo. Fort Myers, Florida. Ehsani, R. and Trotochaud, J. 2015. Technical specifications. Citrus Expo. Fort Myers, Florida. Outside with thermotherapy vehicle. Ehsani, R. and Trotochaud, J. 2015. Typical operation. Citrus Expo. Fort Myers, Florida. Outside with thermotherapy vehicle. We have organized a "thermotherapy workshop" at the 131st Annual Florida State Horticultural Society (FSHS) Conference, in Fort Lauderdale, FL., on June 10-12, 2018. Please see "publication section" for the full list of oral presentations. Example of talks are: Abdulridha J., Ampatzidis Y, Ehsani R., Dai A., Thompson K., Xu Y., 2018. Automated mobile heat thermotherapy system for HLB-infected citrus plants. 131st Annual Florida State Horticultural Society (FSHS) Conference, Fort Lauderdale, FL., June 10-12, 2018. Souri S., Abdulridha J., Ehsani R., Schueller J., and Ampatzidis Y, 2018. The effect of root heat treatment on PMS, LAI, and stomata conductance in HLB infectedtrees. 131st Annual Florida State Horticultural Society (FSHS) Conference, Fort Lauderdale, FL., June 10-12, 2018. Abdulridha J., Ampatzidis Y., Kakarla S.C., and Ehsani R., 2018. Mobile heat thermotherapy system for treating HLB-infected citrus trees utilizing hot water and steam. ASABE Annual International Meeting, July 29 - August 1, Detroit, Michigan, USA. Dai A., Thompson K., Xu Y., Ehsani R., and Ampatzidis Y., 2018. Development and Experiment of a Monitoring System for Steam Treatment of Citrus Greening. ASABE Annual International Meeting, July 29 - August 1, Detroit, Michigan, USA. Duncan, L.W., Fluty, J.Q., El-Borai, F.F.E. 2018. Does management of plant parasitic nematodes FritzRoka, Megan Dewdney, Evan Johnson, Naweena Thapa, Reza Ehsani, Yiannis Ampatzidis, Jaafar Abdulridha. Economic Feasibility of Thermotherapy as a Strategy to Mange HLB Infected Trees. Annual meetings of the Florida State Horticultural Society, Fort Lauderdale, FL. June 13, 2018. Some extension EDIS publications are: Document title EDIS numbera Number distributedb Huanglongbing (HLB; citrus greening) Leaf and Fruit Symptom Identification PP327 4153 Huanglongbing (HLB; Citrus Greening) and Nutrient Deficiency Identification PP328 5440 Citrus Greening (Huanglongbing; HLB) Blight and Tristeza Comparison Identification Sheet PP263 2602 Citrus Greening (Huanglongbing): A Serious Threat to the Florida Citrus Industry CH198 4621 Scouting for Citrus Greening (Huanglongbing; HLB) HS1147 297 Citrus canker & greening (HLB) Handling Protocols for Master Gardener Plant Clinics HS1117 592 The Effects of Huanglongbing on Florida Oranges FSHN11-08 592 TAP Sampling for Asian Citrus Psyllid (ACP) Field Sheet ENY-887 4925 TAP Sampling for Asian Citrus Psyllid (ACP) Field Sheet (Spanish) ENY-890 315 Citrus Pest Identification Sheet -- 4646 aUniversity of Florida Institute of Food and Agriculture Systems (UF/IFAS) Electronic Data Information System (EDIS) bFrom January 2015 to September 2019
Next Reporting Steps
Nothing Reported
Target Audience
Citrus growers, Crop consultants, Agricultural machinery industry. Growers are seeking novel ways to effectively mitigate the negative production effects from citrus greening (i.e. HLB). Early trials using thermotherapy techniques showed promise in suppressing the bacterial effects of HLB and enhancing renewed tree growth and fruit production. This project centered around a large-scale trial where high-temperature steam "cooked" individual trees for varying durations to see if this thermotherapy technique could achieve economic feasibility. If positive trial results were observed, equipment manufacturers would be encouraged to design and build cost-effective treatment systems to apply the thermotherapy treatment.
Changes / Problems
We have struggled to identify an appropriate RNA target for CLas viability in the host. This has delayed our work on the second part of Objective 5 where we planned to calculate the D/Z values for CLas. We are testing another method for RNA primer design and hope to have this resolved in the next month to initiate the experimental work. Hurricane Irma directly affected the trial block. It may not be possible to disentangle hurricane impacts from differences in thermotherapy treatments.
Training & Professional Development
One graduate student and one postdoc have had training opportunities and developed the mobile thermotherapy system and the thermodynamic model. They presented their work in international conferences. One graduate student and one part time undergraduate student continued working on this project year applying the thermal treatments, measuring of chlorophyll fluorescence and participating in the preparation of oral and poster presentations. The undergraduate student gave a local oral presentation at the College's undergraduate research symposium. In part because of the research experience, the undergraduate student is now pursuing a M.S. degree. One graduate student has had training opportunities and has worked on RNA methods and interpretation as well as professional development presenting results at regional and international meetings. Two postdocs have had one on one training opportunities and developed experimental design, data analysis, and presentation skills while studying conditions that induce movement of Candidatus Liberibacter asiaticus between the roots and canopy to better guide application timing. Furthermore, we demonstrate EPN application techniques to grower-cooperators.
Dissemination Streams
The effects of thermal treatments were presented at the Annual International Meeting of the American Institute of Agricultural and Biological Engineers and at the annual meeting of the Florida State Horticultural Society. Furthermore, we demonstrate EPN application techniques to grower-cooperators. Several presentations were given at grower events. Examples are: Y. Ampatzidis. Agricultural engineering technologies to improve citrus production, Citrus Show, Fort Pierce, Florida, Jan-24, 2018. Y. Ampatzidis. Smart Machines, UAV and Precision Farming Technologies, 2018 Citrus Innovation and Technology Show. Arcadia, Florida, April-5, 2018. Y. Ampatzidis. Agricultural and Biological Engineering Technology Applied to Citrus Crops, 2018. Citrus Health Forum, NFREC, Quincy Fl., April 19, 2018. Y. Ampatzidis. New Technologies in Agriculture for Specialty Crops, 2018. Citrus Engineering Conference, Panel Session, Lake Alfred, FL, June 6, 2018. Y. Ampatzidis. Smart and Precision Agriculture, 2018. LaBelle Rotary Club, LaBelle Fl, September 17, 2018. E. G. Johnson. What's going on below ground?. 2018. Citrus Growers' workshop. Sebring, FL. September 25, 2018 E. G. Johnson. Effect of HLB on citrus root density. 2018. Citrus nutrient management for trees affected by HLB. Immokalee, FL. February 2, 2018 We organized a "thermotherapy workshop" at the 131st Annual Florida State Horticultural Society (FSHS) Conference, in Fort Lauderdale, FL., on June 10-12, 2018. Please see "publication section" for the full list of oral presentations. Example of talks are: Abdulridha J., Ampatzidis Y, Ehsani R., Dai A., Thompson K., Xu Y., 2018. Automated mobile heat thermotherapy system for HLB-infected citrus plants. 131st Annual Florida State Horticultural Society (FSHS) Conference, Fort Lauderdale, FL., June 10-12, 2018. Souri S., Abdulridha J., Ehsani R., Schueller J., and Ampatzidis Y, 2018. The effect of root heat treatment on PMS, LAI, and stomata conductance in HLB infectedtrees. 131st Annual Florida State Horticultural Society (FSHS) Conference, Fort Lauderdale, FL., June 10-12, 2018. Abdulridha J., Ampatzidis Y., Kakarla S.C., and Ehsani R., 2018. Mobile heat thermotherapy system for treating HLB-infected citrus trees utilizing hot water and steam. ASABE Annual International Meeting, July 29 - August 1, Detroit, Michigan, USA. Dai A., Thompson K., Xu Y., Ehsani R., and Ampatzidis Y., 2018. Development and Experiment of a Monitoring System for Steam Treatment of Citrus Greening. ASABE Annual International Meeting, July 29 - August 1, Detroit, Michigan, USA. Duncan, L.W., Fluty, J.Q., El-Borai, F.F.E. 2018. Does management of plant parasitic nematodes FritzRoka, Megan Dewdney, Evan Johnson, Naweena Thapa, Reza Ehsani, Yiannis Ampatzidis, Jaafar Abdulridha. Economic Feasibility of Thermotherapy as a Strategy to Mange HLB Infected Trees. Annual meetings of the Florida State Horticultural Society, Fort Lauderdale, FL. June 13, 2018.
Next Reporting Steps
We plan to continue developing the thermodynamic model to better explain the heat therapy's effects and to help equipment designers to further develop the proposed system. We plan to complete root tracings of minirhizotrons at the main field sites to improve our understanding of seasonal and time-temperature combinations effects on root health. While spring treatments have seen an increase in root biomass, minirhizotron scanning and analysis allows quantification of root growth and lifespan. All scans in the first site have been completed and we are near completion in the second field site. Preliminary analysis of traced roots in these scans has been done and a final thorough analysis will be completed by October. Last year, hurricane Irma directly affected the trial block; we want to collect field data during this growing period to further evaluate the heat therapy to treat HLB-infected citrus trees. <br><br>
<br>What was accomplished under these goals? Obj. 1: A new central control box is designed for the mobile thermotherapy system, which is more compact; the connection between the control box and the circuitry is more robust, and its working condition can be better monitored. Ten new water-proof thermal sensors are designed too. Furthermore, a thermodynamic model was developed to simulate the heat distribution inside the tent (canopy cover). Several field experiments were conducted to evaluate the mobile thermotherapy system and the thermodynamic model. Biosensors were utilized in these experiments to evaluate bacterial survival rate after heat treatment. Obj. 2: The third year of post treatment yield was collected from the first field trial site and the second year of yield data was collected from the second experimental site. Although the spring treatment showed promise with recovery of functional root mass after treatment, this was not reflected in fruit yields in year 3. The 2 year recovery in root health also lapsed during this year, showing a regression back to the pretreatment impairment of the root system. Yield in 2015/16, first year after thermotherapy treatment, tree yields were consistently lower and statistically significant from untreated control trees. Yield losses were observed across all levels of thermotherapy treatments and across the three treatment periods. Losses ranged from 2.5 to 5.9 pound-solids per tree. Yield differences across thermotherapy treatments were not statistically different. Treatments ranged from 55 to 60 C and between 0 and 120 seconds of duration. The timing of treatment did not produce significant yield effects. Obj. 3: In 2018, most of the lab work was focused on developing an RNA-based viability assay in response to thermotherapy. Bacterial viability is usually determined by the ability of the bacterial cells to actively grow and form the colonies in media. However, CLas is a fastidious, yet non-culturable bacterium. This makes it impossible to use classical bacteriological techniques to measure the viability of CLas post-treatments. Therefore, a molecular approach to determine CLas viability was tested. The hypothesis of this study is that the RNA expression patterns in CLas changes with the exposure to different levels of heat treatment (different temperature-time combinations). Our overall objective was to develop an RNA-based viability assay for CLas to measure lethality of high temperatures. Along with the goal of developing viability assay, grafting of the treated branches on healthy rootstocks, was also done to evaluate the effect of steam treatment on CLas survival. Comparison of expression pattern of CLas 16S rRNA at different treatment levels of temperature-time combinations showed significant differences amongst the treatments, particularly for 3 hours post treatment (3HPT) samples (p ≤ 0.0005). There is 3.3-fold decrease of CLas 16S rRNA at untreated control (UTC) in 3HPT samples showing RNA degradation under normal conditions. Obj. 4: In the two main field trial sites there was a good distribution of trees with and without damaging Phytophthora root rot prior to treatment. No effects on tree recovery/response nor a consistent response of Phytopthora populations was observed after treatment. Except in the most extreme cases where Phytophthora is already severely damaging the tree is it expected that a Phytophthora dependent response of the tree to thermotherapy would occur. Obj. 5: Nematode management results. Sample intensity was reduced in the second year following thermotherapy to avoid over-sampling the root systems. Sampling occurred 60-75 days after the final spring and fall oxamyl applications. Each sample consisted of 8 cores (2.5 cm dia. X 30 cm depth) per tree which were combined. Nematodes were recovered from the soil by centrifugal floatation and citrus roots were recovered by sieving from a known weight of soil, dried at room temperature and weighed. At the Black, Buffum East and Buffum West sites, ectoparasitic nematode densities in plots treated with oxamyl twice in 2015, 4 times in 2016 and 2017 and three times in 2018 were 96% 80% and 87% less abundant (P<br><b>Publications</b><br>