PHYSIOLOGICAL AND HORTICULTURAL EFFECTS OF BIOTIC CHALLENGES AND ABIOTIC CONDITIONS IN CITRUS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012121
• Project Start Date: Apr 1, 2017
• Project End Date: Mar 31, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Citrus Research and Education Center

Non Technical Summary
Citrus is the most important tree fruit crop globally in terms of volume of production (Figure 1). It is the only major tree fruit crop adapted to the humid subtropical climate of Florida (Syvertsen and Lloyd, 1994). Previous investigations into citrus physiological responses to abiotic conditions yielded production practices that improved citrus growth under various abiotic conditions that limited growth or yield. Currently, citrus production in Florida and other parts of the world has been greatly reduced due to widespread systemic diseases, most notable huanglongbing (HLB), which causes whole plant decline, and in some cases, death. HLB has been shown to interact with limiting abiotic conditions resulting in exacerbated symptoms. These detrimental symptoms can be partially relieved by addressing abiotic factors as demonstrated by (Graham et al. (2014). At the moment, abiotic-biotic stress interactions are focal point of worldwide applied plant science research (Figure 2; Atkinson et al., 2015). This project involves the study of a broad set of physiological approaches intended to improve understanding of citrus abiotic stress responses.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
205	0999	1020	100%

Knowledge Area
205 - Plant Management Systems;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1020 - Physiology;

Keywords

huanglongbing

citrus

source-sink relationships

physiology

Goals / Objectives
ObjectivesAddress the interactions of HLB and abiotic stresses:1. Determine whether shading impacts symptom development of HLB in infected plants.2. Test the effects of water deficit priming on CLas infection transmission.3. Determine whether mineral nutrition can hasten the rate of shoot hardening.4. Soil water and hormonal control of citrus plant flushing cycles and their impacts on CLas transmission by Asian citruspsyllid.

Project Methods
Objective 1. Determine whether shading impacts symptom development of HLB in infected plants.Using PVC tubing, shading nets will be installed over three-year-old CLas infected sweet orange trees in field conditions. Shade treatments will include 50%,30%, 15%, and 0% shade (no shade control). Tree growth and photosynthesis will be measured before installation shade structures, and at 2-month intervals for 2 years. Measurements will include:New shoot flush numbers and lengths.Trunk diameter.Leaf area index.Diurnal curves of photosynthesis and chlorophyll fluorescence.Bloom and fruit set.Fruit drop.Leaf starch content.Root length densityPhloem microscopyObjective 2: Test the effects of water deficit priming on CLas infection transmission.Preliminary study: dry-downThe PI has already demonstrated a method of ascertaining an effective level of soil moisture to induce a priming response (Vincent et al., 2015). This method will be implemented using an automatic irrigation described in (Aguilar et al., 2015) . In this study, gas exchange and chlorophyll fluorescence parameters of potted plants will be measured on a daily basis during a controlled seven-day dry-down process. Gas exchange variables including stomatal conductance and net CO2 assimilation will be measured using an LI-6800 infrared gas analyzer (Li-Cor, Inc., Lincoln, NE), and chlorophyll fluorescence will be measured using a portable chlorophyll fluorometer (Opti-Sciences, Hudson, NH). The target root-zone moisture for priming treatments in the second study will be selected as the zone at which leaf stomatal conductance is reduced but photosynthetic variables are not significantly affected.Priming, Primed acclimation, and HLBHypotheses:Primed acclimation induces SAR similar to that induced by chemical priming.SAR can reduce CLas infection and HLB development.There are genotypic differences in susceptibility and priming response.This study will have 4 factors: rootstock, scion, priming, and challenge. This study will consider two rootstocks (US 892 and Cleopatra) and two scions (Murcott and Valencia sweet orange), with differential susceptibility to HLB. Priming treatments will include an untreated control, a chemically primed treatment (CP), and a PA treatment. Challenge treatments will be either challenge with CLas + ACP or with CLas - ACP. Thus the experiment will be a rootstock (2) x scion (2) x priming (3) x challenge (2) factorial design with 6 repetitions.The PA treatment will consist of 3 weeks of regulated deficit irrigation at the soil moisture level determined by the preliminary experiment. The CP treatment will be an application of hexonic acid according to Llorens et al. (2014) on the same day that the PA treatment ends. The challenge treatments will consist of 1 week in screen cages with 100 psyllids per plant, with either CLas + or CLas - psyllids depending on the treatment. The challenge treatment will begin one week after the end of the PA treatment, giving the PA and CP treatments time to take effect. After the challenge treatment, the plants will be grown for 3 months in a psyllid-free greenhouse to allow CLas infection and HLB symptoms to develop. One reason to include the CP treatment is that it has been demonstrated to be effective in inducing resistance to other pathogens. Thus, if negative results are found with both PA and CP when challenged with CLas, we will repeat the experiment in a reduced fashion, using Alternaria brown spot as the challenge.Variables measuredTo determine treatment effects on overall plant health and metabolism, gas exchange and chlorophyll fluorescence variables will be measured before and after the challenge treatment, and monthly afterward, as well as leaf photosynthetic and non-photosynthetic pigments assayed using a portable leaf spectrometer (CID-Biosciences, Camas, WA). From these same periods, the root and leaf samples will be saved for potential non-targeted metabolomics and phytohormone quantification. At the end of the study, root and stem hydraulic conductance will be determined to assess both priming treatment and infection rate effects on above- and below-ground water relations (Dynamax, Inc, Houston, TX). Subsequently plant leaf, stem, and root tissues will be dried and weighed to assess dry-matter accumulation and allocation effects of treatments. Additionally 10 leaf veins will be sampled per plant for qPCR testing for CLas presence in leaves. New flush production will be quantified and the rate at which new flushes harden will be assessed with penetrometry.Both phytohormone and metabolomics quantification will only be performed on treatment combinations of interest based on the CLas infection rate or other physiological variables. Metabolomic approach will consist of assaying all metabolites using a broad-spectrum GC-MS technique, metabolites noted to be different between treatments will be assessed quantitatively using standards. Phytohormone quantification will be performed according to Hijaz and Killiny (2014).Objective 3. Determine whether mineral nutrition can hasten the rate of shoot hardening.Effects of calcium and nitrogen interactions on the rate of vegetative flush hardening will be tested. First a study will be performed on HLB-free plants in greenhouse. If results are promising, the study will then be repeated in the field on HLB infected and uninfected trees if available. The study will be a factorial of low, recommended, and high levels of Ca and N. The Ca treatments will consist of fertilizing through irrigation water with nutrient solutions containing 50, 100, and 200 ppm Ca. N treatments will consist of solutions containing 600, 1000, and 1400 ppm N in the form of ammonium nitrate. Thus there will be 9 treatments (3 Ca x 3 N) repeated on 6 plants each. Shoot hardening will be tested using a universal penetrometer.Objective 4. Soil water, hormonal control of citrus plant flushing cycles and their impacts on CLas transmission by Asian citrus psyllid.The results of phytohormone ratios from Objective 2 will be used to formulate plant growth regulator mixtures to control flush development. This process will be performed iteratively in which combinations expected to prevent flushing under sufficient water conditions will be tested at various concentrations on plants in greenhouse conditions. When an effective combinations are found, a second mixture will be sought that induces flushing. When the appropriate combination of flush-preventing and flush-inducing plant growth regulators are found to function in greenhouse studies, they will be tested in the field on HLB-free mature plants if available.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:The target audience is citrus growers in Florida and California. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?These results have been presented to growers in local and state-wide grower meetings and in articles in Citrus Industry Magazine, in Science for Citrus Health, and in the relevant academic publications already cited. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We have published 5 publications on this topic, including pest management, photosynthetic biology, carbon transport, and metabolomics of shaded plants. We have found that HLB reduces photosynthesis primarily by reducing leaf area, that not all genotypes are affected equally, however, HLB impacts transport along the length of the transport pathway.Shading reduces numerous disease symptoms and increases early yields of disease infected plants. Kaolin particles films both shade plants, increasing growth, and prevent arrival of Asian citrus psyllid. 2. Nothing to report. 3. Nothing to report. 4. We have learned that mild soil water deficits delay flushing. Defoliation hastens and increases flushing, indicating that flushing responds to a decreased C:N ratio.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: C. Vincent, A. Guha, N. Killiny, and L. Diepenbrock. 2021. Understory environment promotes photosynthetic efficiency and mitigates severity and function of an introduced, vectored pathosystem: a study of a feral citrus population in central Florida. Trees - Structure and Function. 35:1711-1725.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: M.F. Khalid, C. Vincent, R. Morillon, M. A. Anjum, S. Ahmad, and S. Hussain. 2021. Different strategies lead to a common outcome: Different water-deficit scenarios highlight physiological and biochemical strategies of water deficit tolerance in diploid vs. tetraploid Volkamer lemon. Tree Physiology. 41(12):2359-2374.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: S. Welker, M. Pierre, M. Dutt, C. Vincent, and A. Levy. 2021. Phloem transport limitation in Huanglongbing affected sweet orange is dependent on phloem-limited bacteria and callose. Tree Physiology. 42(2): 379-390.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: A. Guha, T. Vharachumu, M. Khalid, M. Keeley, T. Avenson, and C. Vincent. 2021. Short-term warming does not affect intrinsic thermotolerance but induces strong sustaining photoprotection in tropical evergreen citrus genotypes. Plant, Cell & Environment. 45(1):105-120.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: J.H. Suh*, A. Guha*, Z. Wang, S.-y. Li, N. Killiny, C. Vincent, and Y. Wang. 2021. Metabolomic analysis elucidates how shade conditions ameliorate the deleterious effects of greening (huanglongbing) disease in citrus. The Plant Journal. 108(6):1798-1814. *Co-first authors.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: L.M. Mahmoud, A. Shalan, M.S. El-Boray, C.I. Vincent, M. E. El-Kady, J.W. Grosser, M. Dutt. 2021. Application of silicon nanoparticles enhanced oxidative stress tolerance in salt stressed Valencia sweet orange plants. Scientia Horticulturae. 295: 110856.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: M. Pierre, J. Salvatierra-Miranda, M. J. Rivera, E. Exteberria, P. Gonzalez, and C. I. Vincent. 2021. White and red-dyed kaolin particle films reduce Asian citrus psyllid populations, delay huanglongbing infection, and increase citrus growth. Crop Protection. 150: 105792.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience is citrus growers in Florida and California. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?These rsults have been presented to growers in local and state-wide grower meetings and in articles in Citrus Industry Magazine. What do you plan to do during the next reporting period to accomplish the goals?We intend to: 1. Assess the impact of shade on psyllid arrival. 2. Assess the impact of HLB on root:shoot flushing response. 3. Assess the impact of shade on aging and disease development in HLB+ field trees.

Impacts
What was accomplished under these goals? 1. One publication is in review, and two others are in process. We ave found that shade alleviates a number of the metabolic processes associated with HLB-induced stress are alleviated. 30% shade on HLB-affected trees led to 2x the yield as full sun, HLB-affected trees. 50% and 70% shade relieved stress more effectively, but did not enhance yield as much as 30%. We suspect this may be due to increased flowering. We also hypothesize reduced psyllid pressure in the shaded treatments. 2. Nothing to report. 3. We have learned that citrus shoots are more photosynthetically efficient as a whole than are leaves alone, and that HLB effects on shoot morphology are the primary component of the reduction of photosynthesis on HLB+ plants (Keeley et al., MS in review). 4. We have learned that mild soil water deficits delay flushing, but deficits severe-enough to induce some defoliation hasten it. Shade does not change the timing of flushing, but does reduce its intensity. Flushing responds to a clear pattern of root growth in relation to canopy productivity.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Waqar Shafqat(&), Muhammad Jafar Jaskani, Rizwana Maqbool, Waqas Shafqat Chattha, Zulfiqar Ali, Summar Abbas Naqvi, Iqrar A Khan, and Christopher I Vincent. 2021. Heat shock protein and aquaporin expression enhance water conserving behavior of citrus under water deficits and high temperature conditions. Environmental and Experimental Botany. 181: 104270.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Lamiaa Mahmoud, Manjul Dutt, Christopher Vincent, and Jude Grosser. 2020. Salinity-Induced Physiological Responses of Three Putative Salt Tolerant Citrus Rootstocks. Horticulturae. 6/4: 90. https://www.mdpi.com/2311-7524/6/4/90
• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Christopher I. Vincent, Anirban Guha(p), Nabil Killiny, and Lauren Diepenbrock. 2021. Understory environment promotes photosynthetic efficiency and mitigates severity and function of an introduced, vectored pathosystem: a study of a feral citrus population in central Florida. Trees  Structure and Function.
• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Mark Keeley(g), Diane Rowland, and Christopher Vincent. 2021. Citrus photosynthesis and morphology acclimate to phloem-affecting huanglongbing disease at the leaf and shoot levels. Tree Physiology.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience is citrus growers, primarily in Florida, but beginning in 2019, also in California. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?We have published reports for growers on the use pf particle films in both Florida (Citrus Industry Magazine) and California (Science for Citrus Health). Christopher Vincent.2019. Particle films: a growth-enhancing pest management option.Citrus Industry Magazine.October 2019:http://citrusindustry.net/2019/10/02/a-growth-enhancing-pest-management-option/ Christopher Vincent, Monique Rivera, Elizabeth Grafton-Cardwell, and Peggy Lemaux. 2019. Science For Citrus Health. Research Snapshots: Using Particle Films to Manage ACP.https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/Vincent/ What do you plan to do during the next reporting period to accomplish the goals?We will: 1. Publish Natural Area study. 2. Publish kaolin studies. 3. Continue shade studies.

Impacts
What was accomplished under these goals? 1. Studies are underway on shading. We completed a study in a natural area and prepared a manuscript for submission. We implemented shading studies in a field setting as well. Additionally, we oserved improvements in photochemical performance resulting from acclimation to shade. The ongoing field/production setting studies indicate increased yield and improvements in water relations among others. The particle film studies indicate that that red kaolin enhances growth with an increased effect in HLB-infected plants. Manuscripts on all three studies will be submitted in 2020. 2.The natural area study indicates dramatic reductions in the transmission of HLB in the forest environment. 3. Nothing to report. 4. A grower-developed data set will allow us to develop a predictive model for the formation of flush.

Publications

• Type: Book Chapters Status: Published Year Published: 2019 Citation: Christopher Vincent, Raphael Morillon, Vicent Arbona, and Aurelio Gomez-Cadenas. 2019. The Genus Citrus. Citrus in changing environments. ElSevier.
• Type: Book Chapters Status: Published Year Published: 2019 Citation: Vincent, C., Minchin, P.E., Liesche, J., 2019a. Noninvasive Determination of Phloem Transport Speed with Carbon-14 (14 C), in: Phloem. Springer, pp. 153162.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Vincent, C., Rowland, D., Schaffer, B., Bassil, E., Racette, K., Zurweller, B., 2019b. Primed acclimation: A physiological process offers a strategy for more resilient and irrigation-efficient crop production. Plant Science 110240. https://doi.org/10.1016/j.plantsci.2019.110240

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience was citrus producers in Florida. Changes/Problems:Objectives 2 and 3 have been deemed to not be worth pursuing: 2: We found no effect of priming on HLB infection. 3: The method we developed was not effective in distinguishing leaf hardness. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results were reported at the Citrus Research and Development Foundation in an in-person presentation and on the organization's website. A resulting interview was published on Citrus Industry magazines website. Additionally an presentation was given to approximately 300 growers at the annual Citrus Expo. What do you plan to do during the next reporting period to accomplish the goals?1. Continue investigations of mechanisms and growth in the field. Begin shade structure experiments. 4. Continue investigations of internal and environmental controls of flush dynamics.

Impacts
What was accomplished under these goals? 1. We continued to develop new understanding of the response of citrus to different colors of kaolin. Field and potted treatments of red-dyed kaolin have consistently found that red improves growth to an equal or greater degree than the original white kaolin. A student is continuing investigations of the mechanisms of these effects, which we expect are the result of impacts on water relations. 2. Results from water deficit priming treatments were determined to be inconclusive. 3. Our shoot hardening method was not effective, and we have abandoned this line of inquiry. 4. Several experiments were preformed to assess the effects of NAA and GA3 on induction of new flushes. A greenhouse experiment determined that foliar NAA applications at 100-300 ppm and soil applications at 300-1000 ppm were effective in delaying flush by approximately 20 days under flush-inducing conditions. We also observed a pulse-type pattern in the emergence of new flushes, and hypoethesized that this was the reult of age of the preceeding shoot. We conducted a field experiment to determine the effect of bud maturity on efficacy of GA3 to induce new flushes. Buds at 4,6,8, and 10 weeks were treated with GA3. Only buds that recieved GA at 8 weeks induced new flushes. We have interpreted this to mean that a whole plant signal, rather than bud age controls the temporal dynamics of responsiveness. A student is continuing this line of research to assess the causes.

Publications

Progress 04/01/17 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Continue experiments. Publish results.

Impacts
What was accomplished under these goals? Studies addressing Objectives 1 and 4 are in progress using internal funding sources. Are expected in the following reporting period.

Publications