Site Description
This study was conducted in the greenhouse at the University of Florida Institute of Food and Agricultural Sciences (UF/IFAS) Citrus Research and Education Center (CREC) at Lake Alfred, Florida (Latitude 28o5’37” N; Longitude 81o43’30” W) from 2019 to 2021. The study used 2 to 4-year-old Valencia (Citrus sinensis) trees on Kuharske citrange (Citrus sinensis x Poncirus trifoliata) root stock. A total of twenty trees including 10 HLB-affected and 10 healthy trees (hereafter called NHLB) were used. About 76 L-size pots were filled with a potting mix and each tree was transplanted into a pot. The potting mix had compost, perlite, bark, and vermiculite. A potting mix was used because of the complexity (drainage system) of managing large volume of sandy soil in lysimeters, in a controlled environment.
Fertilizer was applied in three splits per year at 135 kg N ha− 1 of calcium nitrate and diammonium phosphate, 67 kg P ha− 1 of diammonium phosphate and 100 kg ha− 1 of potassium sulfate. Other essential nutrients were applied following recommendations by Morgan and Kadyampakeni (2020). Thus, fertilization was done in August and December of 2019; April, August and December in 2020; and April in 2021.
Irrigation water requirement
Each pot was connected to a drip irrigation system with an emitter rate of 4.5 liters per hour and controlled by a timer. To estimate the amount of water for each pot per day, a ten-year average of meteorological parameters such as solar radiation, air temperature, and relative humidity were collected from the Florida Automated Weather Network (FAWN) station that was located 350 m from the greenhouse. Daily reference evapotranspiration (ETo) was calculated from FAWN using Penman-Monteith56 method as described by Zotarelli et al. (2010) using Eq. 1. The calculated ETo was then multiplied average Kc values for healthy trees according to Hamido et al (2017) to estimate daily crop evapotranspiration (ETc) according to Eq. 2 (Table 1). Each pot was covered with much and irrigation events occurred between 7 h and 8 h to minimize surface evaporation.
where ETo = reference evapotranspiration, (mm.d–1), Rn = net radiation at the crop surface (MJ.m–2.d–1), G = soil heat flux density (MJ.m–2.d–1), T = mean daily air temperature at 2 m height (oC), u2 = wind speed at 2 m height (m.s–1), es = saturation vapor pressure (kPa), ea = actual vapor pressure (kPa), es– ea = saturation vapor pressure deficit (kPa), D = slope vapor pressure curve (kPa.oC–1), γ = psychometric constant (kPa.oC–1).
$$ETc=Kc \times \text{E}\text{T}\text{o}$$
2
Where ETc = crop evapotranspiration, (mm.d–1) Kc = crop coefficient, and ETo = reference evapotranspiration (mm.d–1).
Table 1
Estimated monthly citrus water use (ETc) requirement from a 10-year average reference evapotranspiration (ETo). Parameters for ETo estimation retrieved from Florida Automated Weather Network (FAWN) station.
Month
|
2009
|
2010
|
2011
|
2012
|
2013
|
2014
|
2015
|
2016
|
2017
|
2018
|
2019
|
Avg ETo
|
Kc
|
ETc
|
IWR
|
|
--------------------------------------------------- ETo mm/month -----------------------------------------------
|
|
-- mm/month --
|
Jan
|
47.2
|
39.4
|
47.2
|
47.2
|
55.1
|
39.4
|
47.2
|
47.2
|
47.2
|
47.2
|
47.2
|
46.5
|
0.9
|
43
|
39 ± 4.2
|
Feb
|
64
|
49.8
|
64
|
64
|
64
|
64
|
56.9
|
64
|
71.1
|
71.1
|
64
|
63.4
|
0.9
|
56
|
50 ± 5.9
|
Mar
|
86.6
|
78.7
|
94.5
|
102.4
|
78.7
|
86.6
|
94.5
|
94.5
|
94.5
|
94.5
|
86.6
|
90.2
|
0.8
|
74
|
67 ± 7.4
|
Apr
|
121.9
|
121.9
|
137.2
|
121.9
|
106.7
|
114.3
|
121.9
|
121.9
|
121.9
|
121.9
|
114.3
|
120.5
|
0.7
|
80
|
72 ± 7.5
|
May
|
133.9
|
149.6
|
157.5
|
141.7
|
133.9
|
141.7
|
149.6
|
149.6
|
149.6
|
110.2
|
141.7
|
141.7
|
0.8
|
106
|
95 ± 12.7
|
Jun
|
137.2
|
152.4
|
144.8
|
121.9
|
129.5
|
137.2
|
144.8
|
137.2
|
114.3
|
144.8
|
137.2
|
136.5
|
0.8
|
112
|
101 ± 11
|
Jul
|
133.9
|
149.6
|
141.7
|
141.7
|
126
|
141.7
|
133.9
|
157.5
|
133.9
|
133.9
|
141.7
|
139.6
|
0.9
|
131
|
118 ± 8.7
|
Aug
|
133.9
|
126
|
133.9
|
118.1
|
141.7
|
141.7
|
126
|
126
|
133.9
|
133.9
|
118.1
|
130.3
|
0.9
|
113
|
102 ± 8.2
|
Sep
|
106.7
|
114.3
|
114.3
|
106.7
|
106.7
|
106.7
|
106.7
|
114.3
|
106.7
|
114.3
|
114.3
|
110.1
|
1.1
|
120
|
108 ± 4
|
Oct
|
94.5
|
94.5
|
78.7
|
78.7
|
86.6
|
86.6
|
86.6
|
86.6
|
78.7
|
86.6
|
|
85.8
|
0.9
|
81
|
73 ± 5.8
|
Nov
|
53.3
|
53.3
|
53.3
|
53.3
|
53.3
|
53.3
|
61
|
53.3
|
53.3
|
61
|
|
54.9
|
0.9
|
47
|
42 ± 3.2
|
Dec
|
39.4
|
31.5
|
47.2
|
39.4
|
47.2
|
39.4
|
47.2
|
47.2
|
47.2
|
39.4
|
|
42.5
|
0.9
|
37
|
33 ± 5.5
|
Avg ETo is the average reference evapotranspiration from January 2009 to September 2019. |
Kc is the citrus coefficient as described by Hamido et al (2017). |
ETc is the crop water requirement for equivalent to 100% ET. |
IWR is the estimated irrigation water requirement (IWR) assuming 90% irrigation efficiency. For pots subjected to 80% ET, an 80% of the IWR estimated for the 100% ET was calculated. |
Experimental Design
The experiment was conducted in a randomized complete block design. Two irrigation treatments equivalent to a 100% evapotranspiration (ET) and 80% ET to HLB-affected and non HLB-affected (NHLB) trees were applied. Each ET x tree status (HLB-affected or NHLB) combination was replicated 5 times. The treatment structure is presented in Table 2.
Table 2
Treatment structure description for the evaluation of citrus water use dynamics for Huanglongbing (HLB)-affected and non HLB-affected (NHLB) ‘Valencia’ orange trees in Florida.
Treatment
|
Irrigation requirement by crop evapotranspiration (ET, %)
|
Tree status
|
1
|
100
|
HLB
|
2
|
100
|
NHLB
|
3
|
80
|
HLB
|
4
|
80
|
NHLB
|
Meteorological Measurements
An automatic weather station (Davis Pro2, Hayward, CA) was mounted in the greenhouse at a 2 m height to measure weather parameters following procedures by Allen et al (1998). The average solar radiation, minimum and maximum air temperature, mean air temperature and relative humidity (RH) were calculated from the weather station data. Daily reference evapotranspiration (ETo) was calculated for the greenhouse using Hargreaves method as described in Eq. 3 1.
$$ETo=0.023\left(0.408\right)\left(Tmean+17.8\right){(Tmax-Tmin)}^{0.5} Ra$$
3
where Tmax = maximum air temperature (˚C), Tmin = minimum air temperature (˚C), Ra = solar radiation (MJ m− 2), and 0.408 is a factor to convert MJ m− 2 to mm.
Tree Growth Variables
Initial tree height and trunk diameter were measured for each experimental unit before starting irrigation treatment applications. Subsequently, a measuring pole height stick (model 807396 by SOKKIA Corporation, Olathe, KS) and a digital caliper were used to measure tree height and diameter, respectively, every six months at the same location on the trunk until the end of the study. The digital caliper recorded the trunk diameter in the north-south (NS) and east-west (EW) directions of the tree. Tree height and trunk diameter growth were estimated by subtracting the initial before treatment application measurement from subsequent measurements.
Leaf Area Measurement
Initial and final leaf areas were measured using ImageJ, a Java-based image processing program as described by Schneider et al (2012). Twenty fully expanded leaves were randomly selected from each tree and scanned with an HP scanner (HP ScanJet Pro 2500 f1, Palo, CA) and saved as JPEG images. At the time of leaf sampling, a total leaf count for each tree was also done. The saved JPEG images were then imported into the ImageJ application (https://imagej.nih.gov/ij/download.html) where the leaf area was calculated and averaged. The calculated average leaf area was then multiplied by the total leaf count for each tree to estimate total leaf area.
Soil Water Content
Soil water content was measured every 30 minutes for the duration of the experiment by two-pronged capacitance sensors (EC-5, Metergroup, Pulman, WA) connected to EM-50 data logger (Meter group, Pulman, WA). The sensors were installed at 15-cm depth from the surface of the planting medium and 10 cm away from the trunk of the tree in 3 of 5 replicates for each treatment. Average soil moisture content was calculated from the 3-sensors on each treatment.
Stem Water Potential (SWP)
The stem water potential was measured using a portable pressure chamber (Model 1505D, PMS Instrument Company-Albany, OR). The measurements were done using a similar procedure described by Fulton (2014) for higher plants. Four representative leaves per tree (two trees per treatment) were randomly selected and covered with an aluminum foil for 24 h to allow the water potential of the leaves to equilibrate with the water potential of the stem. A sharp razor blade was then used to cut leaf petioles close to the stem and placed into the pressure chamber immediately to avoid any biological and/or physical changes. The chamber was pressurized at 1 Bar/30 s (14.5 PSI) using compressed nitrogen until the discharge of water from the petiole became visible, and the pressure recorded (MPa).
Water Use Dynamics
Water use was determined using sap flow measurements taken on 28 August to 2 September 2020, 26 March to 9 April 2021, and 14 June to 1 July 2021 using the stem heat balance method with an automated flow system using trunk heat balance gauges SGA10, SGA13, and SGB16 connected to data loggers from Dynamax (Flow32 CR1000x and CR1000; Dynamax, Houston, TX). Stem diameters for the measurements ranged from about 10.1 mm to 16.6 mm during the study. A silicon grease was used to improve thermal contact of the gauges to minimize trunk injury. For each treatment, 4 out of 5 trees were used and the sap flow was measured every 30 min for a minimum of a week. The data from the loggers were then converted to water flow per unit diameter size g h− 1 cm− 2. A 24 h daily water flow was calculated for each measuring period and compared among treatments.
Root Growth
Root growth was assessed monthly using transparent acrylic minirhizotrons installed in each plot using methods described by Han et al. (2016). The minirhizotrons were installed either to the east or west of the trunk, along the direction of the drip emitter at 45o angle at 20 cm away from the tree’s trunk to a depth of 50 cm from the surface. The CID-600 root imager (CID-Bioscience, Pullman, WA) was then used to scan roots within the visible area (21 × 19 = 399 cm2) of the minirhizotron to estimate root diameter, length, area, and volume and the results were compared among treatments.
Data Analysis
The two irrigation rates (ET = 100% and ET = 80%) were considered as blocks and tree status (HLB-affected and NHLB) as subplots. Analysis of variance (ANOVA), using the generalized linear mixed model procedure (PROC GLIMMIX) as implemented in SAS (SAS/STAT 15.1, SAS Institute, Cary, NC [2018]) was used to analyze all response data. When significant (at α = 0.05), a multiple comparison by Tukey’s post hoc honest significance difference test was performed. Correlations and linear regression between variables were determined using Sigma Plot software (version 12.3; Systat Software Inc, San Jose, CA). An unstructured covariance model (UN) was chosen as a best fit to model the repeated nature of some parameters based on Akaike’s Information Criterion corrected for small sample size (AICC). Response variable measured at the end of the experiment was analyze based on a complete factorial combination of treatment factors irrigation rate and tree status. Visual inspection of residuals 17 indicated no violations of the underlying assumptions.