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horticulturae
Review
Sap Analysis: A Powerful Tool for Monitoring Plant Nutrition
Eduardo Esteves 1, Guilherme Locatelli 1, Neus Alcon Bou 1and Rhuanito Soranz Ferrarezi 2, *
Citation: Esteves, E.; Locatelli, G.;
Bou, N.A.; Ferrarezi, R.S. Sap
Analysis: A Powerful Tool for
Monitoring Plant Nutrition.
Horticulturae 2021,7, 426. https://
doi.org/10.3390/horticulturae7110426
Academic Editor: Moreno Toselli
Received: 29 August 2021
Accepted: 20 October 2021
Published: 22 October 2021
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1Indian River Research and Education Center, University of Florida, 2199 S Rock Road, Fort Pierce,
FL 34945, USA; eduardo.esteves@ufl.edu (E.E.); locatellig@ufl.edu (G.L.); neusalconbou@ufl.edu (N.A.B.)
2Department of Horticulture, University of Georgia, 1111 Plant Sciences Bldg, Athens, GA 30602, USA
*Correspondence: ferrarezi@uga.edu; Tel.: +1-706-542-2471
Abstract:
Horticultural crop production is moving towards an era of higher nutrient use efficiency
since nutrient deficiencies can reduce plant growth, productivity, and quality, and overfertilization
can cause environmental pollution. Rapid nutrient concentration diagnostic is essential to minimize
the negative effects of Huanglongbing (HLB) or citrus greening in citrus by providing the required
nutrients before deficiency symptoms appear, reducing the impact of the disease on crop production.
Sap analysis is an additional tool for fine-tuning nutrient applications in citrus. The main objective of
this paper is to review the different methodologies and results obtained with sap analysis, considering
its potential application in citrus production. Results from other crops show the pros and cons of
using this tool. Substantial research has been conducted on vegetables and greenhouse crops, but few
studies are available on perennial species such as citrus. Inconsistency in the extraction and analysis
methods and the lack of specific sufficiency ranges for citrus open the path for further studies. Along
with soil and leaf analyses, sap analysis is a complementary technique that can improve nutrient
use efficiency in citrus production. Moreover, sap analysis has the potential to optimize fertilizer
application, minimize environmental impacts and improve sustainability.
Keywords:
nutrient analysis methods; fertilizer application; nutrient use efficiency; nutrient loss;
fertilizer management; controlled environment agriculture
1. Introduction
Horticultural crops such as fruits and vegetables require optimized irrigation and
fertilization strategies to achieve high yield and quality [
1
–
4
]. Enhanced nutrition is a
viable strategy to keep citrus (Citrus spp.) trees productive and the growers in busi-
ness in the Huanglongbing (HLB) or citrus greening era [
5
–
11
]. However, some grow-
ers are applying more nutrients than needed to compensate for the negative effects of
HLB
[12–16]
. An excessive fertilization strategy can reduce profitability and damage the
environment due to groundwater contamination, eutrophication, and change in microbial
dynamics
[2,12,17–19].
Citrus production and agriculture in general are moving towards
more precise nutrient management, where optimized and more efficient techniques are
taking place [
3
,
10
,
20
,
21
]. To optimize citrus nutrition and nutrient supply, it is essential to
understand the crop nutrient requirements and have real-time diagnostic tools to determine
the current nutrient status inside the plant. In this scenario, leaf and soil nutrient analysis
are standard tools to assess the nutrient status of citrus trees [
14
,
22
–
24
], but the nutrients
contained in the leaf tissue may reflect an accumulation during the plant’s entire cycle or
season, rather than indicating the real-time concentration that is available for plant devel-
opment, especially with elements such as Ca and B, which are unlikely to be remobilized
once they are incorporated into the plant tissue [
25
–
27
]. This also applies to elements such
as N, which may need more sensitive methods to determine real-time changes [
2
,
19
,
27
–
30
].
In this scenario, more precise monitoring tools and techniques are required.
Plant sap analysis is an option for determining plant nutrient status. Some authors
define sap as the liquid portion extracted from xylem and phloem, plus the apoplastic,
Horticulturae 2021,7, 426. https://doi.org/10.3390/horticulturae7110426 https://www.mdpi.com/journal/horticulturae
Horticulturae 2021,7, 426 2 of 13
cytosolic, and vascular fluids [
19
,
29
–
31
], although there is no consensus yet in the scientific
community about this definition. Researchers consider sap as fluids from conductive
tissues [
26
], either xylem, phloem, or a mix [
19
,
29
–
31
]; others describe sap as the xylem
fluids [
32
–
34
]; and several consider sap as just the phloem fluids obtained by insect stylec-
tomy [
35
–
37
]. Nevertheless, the nutrients found in sap are readily available for the plant’s
development [26,28]; therefore, sap analysis is compared as a tree “blood test”.
Plant sap analysis provides an early determination of the plant nutrient status since it
relies on real-time information [
1
,
28
,
29
,
38
–
40
]. Plant mineral levels, nutritional deficiencies,
and excesses could be determined before they cause any damage to plant development and
consequently fruit yield [
26
,
28
]. Different sap analysis methods are available, and some
private companies and commercial laboratories compare sap of new vs. old leaves. In
addition to the regular macro and micronutrient indicators that leaf analysis provides, some
laboratories include NO
3−
-N and sugar content in their reports. These two parameters
can provide information on the plant metabolism, if N is being transformed rapidly into
proteins, or if there are high levels of soluble N resulting in increased water uptake and
dilution of sugar levels, which could increase pest and disease attack [
11
,
41
,
42
]. Further-
more, sap analysis provides the opportunity for growers to adjust fertilization and apply
the specific amount of nutrients needed, not only for plant nutrition but also for improving
environmental sustainability.
The first reports and attempts to study the effects of fertilization on sap composition
were performed in the U.S. by Dr. Pettinger and Dr. Arnon in the 1930s [
43
,
44
]. The early
publications on measuring and interpreting plant sap dates were generated in Europe
in the 1970s [
25
,
45
]. In Florida, there is vast experience with sap analysis, especially for
vegetable and greenhouse crops. Dr. George Hochmuth (Emeritus Professor, University of
Florida) conducted numerous sap testing and interpretation studies, emphasizing portable
devices and quick testing [31,46–48].
In recent years, plant sap analysis is receiving more attention in citrus [
43
,
44
] because
it can assess plant nutrient uptake more precisely, increase fertilizer efficiency, reduce envi-
ronmental constraints, enhance fruit quality, and improve disease management
[1,26,40,49]
.
The analysis is not considered an alternative to leaf analysis but a complementary tool for
nutrient and disease management [
1
,
19
,
26
,
38
–
40
,
50
]. Research indicates that HLB-affected
citrus trees have lower nutrient concentrations in leaves than healthy trees
[6,44,48,49]
.
Sap analysis can rapidly determine nutrient deficiencies and guide the application of the
required nutrient accordingly during each phenological stage.
However, sap analysis has its limitations. The availability of different equipment
and methodologies introduces variabilities and inaccuracies to the results, reducing the
reliability of the information [
1
,
50
]. According to [
51
], there is a gap between sample
collection, chemical analysis, and nutrient supplementation in sap analysis. Future research
should standardize the sampling and extraction methodology, establish reference levels for
each nutrient, and develop correlations with yield and fruit quality variables. Some private
companies and laboratories have developed sufficiency ranges and interpretation charts
for some crops; however, many of these laboratories do not disclose their methods and/or
reference levels, making it harder for growers and scientists to compare results. This is
critical for sap analysis since the results are affected by different factors. A large portion of
the studies focused mainly on N and greenhouse crops [
2
,
28
,
29
,
47
]. Still, little research has
been conducted with micronutrients, which seem to alleviate the effect of plant diseases
such as HLB in citrus [6,15,16].
Our objective with this publication is to review the different methodologies and results
obtained with sap analysis, considering the potential application of this nutrient manage-
ment technique in citrus. Additionally, we suggest some research ideas, as sap analysis
could become another tool for improving citrus nutrition and nutrient use efficiency. If
plant sap analysis is combined with soil and leaf analysis as a management tool, growers
will have access to a more robust approach to assess citrus nutrition and address many
Horticulturae 2021,7, 426 3 of 13
current and future challenges, increasing fruit yield and juice quality, enhancing fertilizer
application, increasing revenue, and reducing environmental impacts.
2. Procedures for Sap Analysis
Plant sap analysis is an operationally defined method, meaning that the analysis
results will highly depend on the chosen methodology since it has not been standardized.
There is still no consensus among the scientific community regarding a unique sap analysis
methodology for sample collection, tissue type (petioles, shoot tips, and leaf blades),
pressing equipment, sap extraction, or fluids analyses [
19
,
27
,
28
,
34
,
47
]. Therefore, our
goal is to describe the different definitions and methodologies involved in sap analysis so
that readers and the scientific community can have a baseline to start defining a general,
standardized, and consented methodology. There are three main steps in the sap analysis:
sample collection, sap extraction, and sap analysis (Figure 1).
Figure 1.
Sap analysis methodologies: sample collection, sample extraction, and sample analysis. Procedures inside each
stage are not necessarily a sequence but different approaches used in several studies.
2.1. Sample Collection
The sample collection is a critical activity that requires specific considerations. The
sampling strategy must consider and separate potential differences typically found in
groves, such as soil types, cultivars, and management practices [
26
]. The samples should be
taken at a similar stage within the same group of well-watered trees because sap nutrient
concentration may vary depending on the crop stage, some of them declining with the
growth stage and time [
26
,
27
,
39
,
49
,
52
]. At the sample collection, we should consider the
type of tissue and timing.
Horticulturae 2021,7, 426 4 of 13
2.1.1. Type of Tissue
The type of tissue sampled might impact the results obtained [
1
,
38
–
40
,
50
]. Most
authors have used petioles as the sampled tissue, usually taking the petioles from the
most recent fully expanded leaf [
2
,
19
,
28
–
30
,
34
,
40
,
53
,
54
]. Instead of using petioles, in [
55
]
and [
56
], the leaf blade midribs were used for sap analysis in broccoli (Brassica oleracea)
and sugar cane (Saccharum officinarum), respectively, while in [
52
], the use of leaf blade vs.
petioles was compared for sap analysis in strawberries (Fragaria
×
ananassa). In [
57
–
59
], leaf
blades for sap analysis was recommended, which is becoming an interesting adaptation of
the method by private companies in the Netherlands [19].
There are different approaches depending on the crop to be sampled regarding the
number of leaves/petioles for each sampling unit. For potatoes (Solanum tuberosum) and
tomatoes (Lycopersicon esculentum), some studies reported around 20–25 leaves and petioles
from the most recent fully expanded leaves [
29
,
40
,
60
]. In strawberries, researchers have
reported the need for 60 to 100 leaves and petioles [
52
,
53
,
59
], while grapevines (Vitis spp.)
may require about 200 [
61
]. The number of tissue samples may also be a function of
the nutrient to be measured and the methodology. [
30
] reported the need for 22, 3, and
113 tomato petioles when analyzing NO
3−
-N, Cl
−
and H
2
PO
4−
, respectively. The number
of leaves/petioles for each sampling unit might be a function of different factors, including
site-specific conditions. We propose collecting 30 to 60 whole citrus leaves (including
petioles) for extracting enough sap for one sample—suggesting no more than three leaves
per tree—and a separate analysis of each sample. This coincides with the methodology
used by [
57
] and [
58
], who took 40 leaves when working with sweet orange (Citrus sinensis)
cultivars.
Nowadays, some commercial laboratories offer an analysis comparing old to new
growth, especially for a nutrient mobility assessment. In citrus, an old leaf is consid-
ered a dark-green, active leaf and distant from the growing point. A new leaf would be
fully expanded, but from the latest flush, located close to the growing point and with
a light green color. Few authors have followed this approach of collecting old and new
growth [
58
]. Most of the published work related to sap analysis has been focused on
N and mostly in greenhouse crops, taking the most recent fully expanded leaves and
petioles
[2,19,28,29,39,40,53,55,56,60]
. It is well known that N is a mobile element inside
the plant, moving from old to new growth. These are probably valid reasons why most
published work has not compared old and new growth results. However, in [
29
], the
limitation of sap analysis to show a decrease in plant N accumulation later in the crop
cycle if the petioles are always collected from the top part of the plant (new growth) is
highlighted. Furthermore, N should not be the only element of interest in sap analysis, as
there are other essential nutrients with low mobility inside the plant, such as Ca and B [
62
],
which may benefit from an old vs. new growth comparison. The nutrient assessment of
perennial crops could be improved with this perspective.
2.1.2. Timing and Frequency
Consistency is a critical aspect of plant sap sampling since both the time of day
and the frequency should remain constant for comparing the results. The time of day
is an essential factor, as nutrient concentrations may vary throughout the day. In wheat
(Triticum aestivum), sampling before and after 2 pm showed a 10% and 40% difference
for K and Fe sap concentrations, respectively, with higher values in the afternoon [
35
].
In another experiment with tomatoes, higher NO
3−
-N, NH
4+
, and H
2
PO
4−
-P sap values
were also found in the afternoon [
34
]. However, in ‘Sultana’ grapevines, K levels were
50% lower in the afternoon [
61
]. In potato, sap NO
3−
-N levels tended to increase at
noon and mid-afternoon, decreasing later at night [
60
]. As the timing for collecting sap
samples has also been inconsistent among different studies, some methodologies suggested
collecting leaves before 10 am (generally between 7 and 10 am) in crops such as sweet
peppers (Capsicum annuum) and broccoli [
2
,
28
,
55
]. In contrast, others preferred tomato
leaves to be collected from 10 to noon [
29
] or even in the afternoon [
62
]. The fluctuations
Horticulturae 2021,7, 426 5 of 13
in nutrient concentration are probably associated with leaf water potential variations;
therefore, morning hours may be suggested for sap sampling, as this would minimize
variability [60].
The sampling frequency is another factor to consider. Results have indicated that sap
N levels may remain constant during the crop cycle, suggesting that sampling could be
carried out just once during a crop cycle. When working with sweet pepper in greenhouse
conditions, petiole sap NO
3−
-N content remained relatively stable throughout the crop
cycle [
2
]. Similar results were found in muskmelon (Cucumis melo) and tomatoes [
39
].
However, when dealing with open field conditions, the nutrient levels may increase or
decrease depending on the crop stage, as supported by [
26
,
31
,
52
]. Frequent and low
N dosing, combined with fertigation and drip irrigation, may contribute to a constant
petiole sap NO
3−
-N content through the crop cycle in greenhouse conditions [
2
,
19
,
39
,
49
].
According to [
29
], the sap test could show steady N concentrations because the petioles
are always collected from the top of the plants (new growth), forcing samples to be taken
from old and new growth. Therefore, it may be inferred that perennial crops in open
field conditions may require more than one sampling per season. For citrus, the sampling
frequency would depend on the market and the variety. Fruit quality monitoring for the
fresh industry, e.g., mandarins (Citrus reticulata), grapefruit (Citrus
×
paradisi), and sweet
oranges, would require more frequent sampling throughout the season.
2.2. Sample Extraction
After the samples have been collected, they should be kept cool, prevented from
desiccation, and processed within the first 24 h to avoid degradation, leading to inaccurate
results and wrong interpretations [26].
Sampled tissue could be sliced into 0.5 cm pieces, submerged into ether (98% v/v), and
put into a freezer for at least 2 h [
25
]. The rationale for freezing is to crystalize the tissue
and help obtain the fluids in the latter pressing, as NO
3−
-N and K release increased when
petioles were frozen [
61
]. As chlorophyll could interfere with the analysis, the ether is used
for sap extraction. Later, the sample is defrosted, and the ether and chlorophyll solution
(green colored fluid) is separated from the sap by a funnel. This methodology was followed
by [
27
,
30
,
56
–
58
]. Some authors also froze and defrosted tissues before pressing, but they
did not mention the use of ether in their methods [
28
,
53
]. Other studies treated their
samples without freezing and conducted pressing/crushing immediately [19,31,54,55,60].
When pressing/crushing is part of the methodology, the press/crusher should be
made of PVC, stainless steel, or even nylon to avoid cross-contamination with metallic
elements [
26
]. While using petioles as the sampled tissue, some authors sliced the petioles
in 5–10 mm pieces and then pressed tomato and sweet pepper tissues in a stainless steel
garlic crusher [
19
,
28
,
54
]. A similar methodology was followed by [
2
], collecting larger
petioles (1 cm slices) in sweet pepper and using a garlic press for sap analysis. However,
cutting and/or washing pieces of petioles may reduce the N and K sap concentrations
in muskmelon and sweet pepper than pressing the whole petiole [
54
], which shows the
importance of standardizing the sap sample extraction. Instead of using a garlic press,
other studies used a hydraulic press for crushing the tissues [
26
,
27
,
30
]. Besides press-
ing/crushing, other interesting methods include using a Pasteur pipette for collecting
sap [
34
] or using aphid stylectomy to obtain the fluid [
35
,
63
]. The authors of this review
have tried pressing citrus leaf petioles and blades with a garlic press without success. The
garlic crusher seems to be more effective with leaves and petioles that are ‘fleshier’, such
as tomatoes, sweet peppers, or potatoes; however, citrus leaves might require a hydraulic
press or another type of extraction. It would be important to quantify and set a standard
pressure for citrus leaves to standardize the methodology.
2.3. Sample Analysis
The plant sap analysis could be performed by a laboratory with specialized equipment
or by the user/grower with portable devices. Nevertheless, before any analysis, a dilution
Horticulturae 2021,7, 426 6 of 13
may be required. Typically, the sap is diluted because the nutrient concentration exceeds
the measurement range of the device [
49
,
50
], but also the green chlorophyll color may
interfere with the measurement of colorimetric devices [
48
]. A compilation of different
dilution ratios for each nutrient is listed in Table 1.
Table 1. Dilution ratios used in different studies for several types of analyses.
Nutrients Analyzed Solvent Ratio Type of Analysis Authors
NO
3−
-N, NH
4+
, P, B, Ca, K, Mg, and Na
HCl 2% 1:25 Spectrometry
[26]
Fe, Cu, Mn, and Zn HCl 2% 1:10 Spectrometry
Cl−HCl 2% 1:25 Ion selective electrode
Total N - - Kjeldahl method
NO3−-N Deionized water 1:200 Colorimetry [28]
K Deionized water 1:20 Spectrometry
NO3−-N Distilled water 1:20 Strips and reader [55]
NO3−-N and K Distilled/deionized water 1:50 Strips and reader,
colorimetry, and electrodes [48]
Portable devices are usually a faster and cheaper method for obtaining results [
31
,
40
,
64
]. When using some ion-selective strips, a color reagent is added to the pressed sap,
and the color is compared with a standard chart color that indicates different levels (low,
medium, and high) [
62
]. These strips could also be analyzed with a reader based on
reflectometry, which upgrades the method from semiquantitative to quantitative [
48
,
65
].
Around 1990, a battery-operated handheld ion-selective electrode was introduced, which
directly measured sap without the need for dilutions and/or color reagents [
50
]. With
these portable devices, many reference levels and sufficiency ranges were developed. The
University of Florida has used petiole-sap testing for vegetable crops in Florida with mobile
devices. Studies include N and K sufficiency ranges for tomatoes, sweet peppers, straw-
berries, and watermelons (Citrullus lanatus), but not for citrus [
31
,
47
]. Some publications
compile and describe the handheld devices available for measuring petiole sap NO
3−
-N
in potatoes, including their brand names, pros, and cons [
40
]. The accuracy of a portable
ion-selective electrode was compared to a laboratory method for sap NO
3−
-N analysis. The
studies concluded that this device was sufficiently accurate to guide on-farm decisions [
66
].
However, other authors suggest using strips instead of electrodes for NO
3−
-N evaluation
in vegetables [
65
]. This portable equipment could give real-time and on-site data; however,
they have limitations. For example, according to [
50
], fouling the ion-selective membrane of
an electrode meter can cause inaccuracies that would add more limitations to sap analysis.
Moreover, organic compounds and ions such as Cl
−
could interfere with the electrode
measurement, reducing the accuracy [
65
]. Likewise, when using test strips, it is possible
that the high dilution rate, in addition to other ions or substances, may affect the re-
sults [
61
,
66
]. These quick analyses should be used carefully, with results compared against
laboratory check analysis and using equipment calibrated and serviced regularly [49,66].
On the other hand, there are several non-portable methods for analyzing the sap
extract (Table 1). While in [
26
,
27
,
34
], atomic absorption spectrophotometry and [
30
] used
high-performance liquid chromatography were used, others [
56
] used the Kjeldahl method
for inorganic forms of N and sulfuric digestion and distillation for the rest of the nutrients.
A plasma spectrometer has also been used to analyze sap in citrus [
43
], while in [
2
,
33
], a
continuous segmented flow analyzer was used to measure sap levels from tomato and
sweet pepper, respectively.
The vast range of methods for each step is evident. The differences in methodologies
make it more challenging when interpreting results, developing reference levels, and
spreading the concept among users/growers. The accuracy and precision may differ from
method to method and the turnaround time for obtaining the results.
Horticulturae 2021,7, 426 7 of 13
3. Sap as a Potential Nutrition Index for Citrus
An adequate fertilizer application requires knowledge of the crop’s nutrient require-
ment. Soil and leaf analyses are needed to develop a nutrient management plan and follow
the best management practices [
62
]. However, the nutrient concentrations in the crop tissue
and the interpretation of results may differ from crop to crop, even among cultivars within
the same crop.
Studies have measured sap nutrient levels for ‘Valencia’ and ‘Hamlin’ sweet oranges,
and the results are shown in Table 2[
57
]. These are not meant to be sufficiency ranges but
just an idea of how citrus sap nutrient levels vary. For example, citrus sap NO
3−
-N values
may be lower compared to other crops. Some vegetables, such as pepper or eggplant,
have NO
3−
-N reference levels above 1000 mg L
−1
[
2
,
26
,
31
], while in [
58
], 223 mg L
−1
was
reported as the highest value in their study with ‘Pera’ sweet oranges. According to [
57
],
NO
3−
-N represents no more than 5% of the total N in citrus, and this could happen because
citrus has a high NO
3−
-N reduction rate. Therefore, higher NO
3−
-N values in citrus sap
could indicate health or metabolic issues.
Table 2.
Sap nutrient concentration for control treatments in ‘Valencia’ and ‘Hamlin’ sweet oranges (Citrus sinensis). Adapted
from [57].
Cultivar pH NH4+NO3−-N Total N P K Ca Mg S B Cu Fe Mn Zn
Sap Nutrient Concentration (mg L−1)
‘Valencia’
5.4 23.6 62.8 86.4 3600 4000 596.8 474.4 156.8 4.0 2.1 1.7 0.9 2.6
‘Hamlin’
5.5 22.8 61.6 84.4 3500 3800 581.8 468.5 139.4 3.6 2.1 1.3 0.9 2.4
Sap nutrient concentration could be a function of many factors, such as sampling
stage and cultivar. The crop sampling stage may affect sap P levels, as these are reduced
after fruit set in nectarines (Prunus persica var. nucipersica) and some vegetables [
26
]. This
finding is also supported by [
30
], who found that sap P levels in tomatoes had a coefficient
of variation of 71% through the crop cycle, compared to 9% for K and 11% for NO
3−
-N.
This suggests that the sap P levels may vary significantly through the crop cycle, even in
controlled environmental conditions. Moreover, when sampling different cultivars from
the same crop, substantial differences may arise. In sweet orange cultivars, sap P levels
could vary considerably, as in [
57
], the presented P sap values were ten times higher in
‘Pera’ oranges than ‘Hamlin’ and ‘Valencia’ [
58
], even when both experiments followed
similar methods. In addition, sap P levels were affected by P fertilization treatments in the
‘Valencia’ cultivar but not in ‘Hamlin’ [57], suggesting the strong influence of the cultivar.
In nutrient assessment, sap analysis could be a more sensitive tool than leaf analysis in
citrus. When supplying Zn and Mn as fertilizers to ‘Pera’ sweet orange trees, in [
58
], a 2-fold
increase with Zn and a 3-fold increase with Mn in leaf nutrient concentrations were found
with leaf analysis. However, with sap analysis, they found a 5-fold increase with both Zn
and Mn. Sap analysis could also indicate interactions that may be hidden in the leaf analysis.
Researchers obtained significantly lower sap P levels with a Zn fertilization treatment when
compared to Mn fertilization in ‘Pera’ sweet oranges [
58
]. This could be explained by the
well-known negative interaction between Zn and P [62]. When checking correlations, sap
NO
3−
-N was negatively correlated with both sap Cu (
−
0.93) and leaf Cu (
−
0.91) [
58
]. The
other correlations between leaf and sap nutrients were not significant (p> 0.05), which
supports the idea that leaf analysis could indicate the nutrient accumulation, while sap
analysis could provide the real-time nutrient availability inside the plant. Nevertheless,
research is still needed for considering sap analysis as a supplemental tool for nutrient
management in citrus, especially when looking for reference levels and understanding how
these levels are influenced by different types of soil, climate, and management.
Limited research has been published in citrus sap analysis, especially related to result
interpretation. Further studies should establish sufficiency ranges for sap measurements in
citrus (both HLB-affected and non-affected) to allow precise crop production since there
Horticulturae 2021,7, 426 8 of 13
is the potential for optimizing fertilizer application by interpreting data from plant sap
analysis. Citrus nutrient management can be improved significantly by combining soil test,
leaf, and plant sap analysis.
4. Sap as a Nutrition Index for Other Crops
Unlike citrus, sap analysis has been studied in vegetable crops and some perennials
in recent years. Many studies have focused on optimizing crop N management since this
technique is susceptible to NO
3−
-N changes in the crop [
29
,
39
,
40
,
53
,
56
]. However, the
materials and methods varied with each experiment.
4.1. Vegetables
Tomato is probably the crop with the highest number of publications related to
sap analysis. Most of these studies aimed to fine-tuning N fertilization in controlled
environments. In a fertilization experiment with different N rates, in [
29
], the N rate
and the type of fertigation and irrigation systems affected the sap NO3−-N concentration.
Similar results were obtained by [
55
] with broccoli. The authors reported that sap analyses
successfully assessed crop N status, creating a management tool for N fertilization.
The different fertilization rates or the irrigation system could influence the sap values
and the soil or substrate used to sustain the crop. Lower P sap concentrations were found in
tomatoes when grown in a soil and sand substrate compared to Rockwool [
26
]. Apparently,
P fixations/reactions in the soil caused the lower sap P levels, as these reactions did not
occur in the Rockwool. One of the most interesting findings in the same experiment was
the competition between NO
3−
-N vs. Cl
−
and Ca
2+
vs. Mg
2+
at the sap level, meaning that
the supply of one of these nutrients could impair the uptake of the other and vice versa.
This finding is also supported by other authors [30,67].
Sufficiency levels may not be easy to define and might require taking several samples
from different cultivars, soils, management regimes, etc. Nowadays, there are emerging
methods for determining sufficiency values. Studies have determined N reference values
by equations describing the relationship between petiole sap NO
3−
-N and the Nitrogen
Nutrition Index (NNI) in crops such as tomato, muskmelon, and sweet pepper. To calculate
NNI, a critical N curve related to the dry weight of the crop is needed [
2
,
39
]. As a reference
for vegetable sap nutrient values, sap sufficiency levels for two tomato crop stages are
compiled in Table 3.
Table 3.
Sap nutrient concentration for tomato (Lycopersicon esculentum) throughout the crop cycle and at harvest. Adapted
from [30,31].
Crop Stage Sap Nutrient Concentration (mg L−1)Authors
NO3−-N H2PO4−-P K+Ca2+ Mg2+ Na+Cl−
Throughout the crop cycle 1253 39.5 4533 555 1688 5512 3120 [30]
Harvest 700 - 3500 - - - - [31]
The N accumulation in tomato biomass was highly correlated with the petiole sap
NO
3−
-N concentration in the leaves during the crop cycle [
29
]. Moreover, the sap NO
3−
-N
results with portable devices have matched laboratory analyses across the full range of
NO
3−
-N concentrations examined. Therefore, studies concluded that sap analysis is a
practical method to assess crop N status, and petiole sap NO
3−
-N is preferable to leaf
N content as it gives a real-time assessment of crop N status and can be analyzed with
quick on-site tests. However, high sap NO
3−
-N concentrations could result from NO
3−
-N
excess in the soil solution due to the high N supply at a specific event or time point [
30
,
39
].
If these results are not contrasted with other analytical methods like leaf analysis, they
could provide a misleading interpretation of excess N in the crop. Thus, the importance
of keeping both leaf and sap analysis as complementary tools for nutrient assessment is
highlighted.
Horticulturae 2021,7, 426 9 of 13
Sap analysis has also been evaluated in potatoes, especially for N nutrition, as some
researchers found it highly correlated with the rate of N-fertilizer applied [
60
,
64
]. Other
studies have compared different methods for N assessment, including sap analysis and
chlorophyll meters. The chlorophyll meters tend to indicate the N assimilation; however,
they do not detect luxury N consumption in potatoes, as opposed to the sap analysis [
40
].
Moreover, the sap analysis seems to be a more sensitive tool to differentiate fertilization
rates at different stages [
51
]. Even though sap analysis results are highly dependent on
external factors (cultivar, soil, fertilizer supply, and weather), sap analysis seems to be
a more accurate method to assess N status in potatoes than chlorophyll meters [
40
,
65
].
Additionally, sap analysis provides a more holistic assessment in terms of plant nutrition.
4.2. Strawberry
Sap analysis has been studied extensively in strawberries. In [59], authors correlated
dry leaf weight and leaf sap, and found that sap NO
3−
-N was not significantly corre-
lated with leaf NO
3−
-N, and the same result was found for Cl
−
, B, Zn, and S. It is not
surprising that leaf and sap NO
3−
-N are not correlated, as the NO
3−
-N is rapidly reduced
and transformed into proteins, once is taken up by plants [
68
]. NH
4+
, P, K, Mg, Ca, Fe,
Mn, and Cu were significantly correlated. However, B and Zn may not be correlated
due to their low mobility inside the plant [
62
], allowing sap analysis to assess immobile
nutrients more accurately. Strawberry reference levels from different authors are shown in
Table 4. Although some values are in a similar range, others may differ due to different
methodologies and/or cultivars.
Table 4. Reference levels for leaf petiole sap in strawberries (Fragaria ×ananassa).
Crop Stage Sap Nutrient Concentration (mg L−1)
NO3−-N P K+Ca2+ Mg2+ Na+Cl−Authors
Blooming summer 350–500 295–425 4500–5000 850–1000 300–450 40–50 - [69]
Fruit set summer 600–800 140–210 4300–4800 450–600 200–300 30–40 500
March 500–700 250–360 4200–5600 700–1200 300–610 - 500–780 [26]
May 300–550 220–330 4200–5800 500–610 190–310 - 330–500
March 200–500 - 1800–2500 - - - - [31]
April 200–500 - 1500–2000 - - - -
When interpreting sap analysis results, it is advisable to look for possible interactions
among nutrients. As mentioned previously, Cl
−
vs. NO
3−
-N is a good example, as there is
an interaction in which a reduced NO
3−
-N uptake takes place when high amounts of Cl
−
are available in the soil [
26
,
30
,
67
]. This is important because a nutrition approach using
either water or fertilizers high in Cl
−
could lead to N deficiencies in the crop [
67
]. Another
interesting interaction occurs between K and Ca. In [
70
], a strawberry trial was conducted
in Spain from November to May, applying three different soil preplant treatments: NPK,
NPK + manure, and NPK + manure + gypsum + dolomite. Leaf and sap samples were
collected for analysis at 8,12, 19, and 23 weeks after planting. The sap results showed an
interaction between K and Ca, as the treatment having no Ca (NPK) had higher K sap
levels when compared to the other two treatments. Sap analysis could become a valuable
tool for tracking fruit quality as the K:Ca ratio influences fruit quality in strawberries [
71
].
4.3. Grapevine
Another crop studied regarding sap analysis and nutritional diagnosis methods is
grapevine. After working with sap analysis in different fertilization levels, in [
26
], specific
nutrient guidelines were defined for sap in grapevine (Table 5). One-year-old plants of
Vitis vinifera ‘Red Globe’ were grown with three different increasing fertilization treatments:
N (0, 2.56, 5.12, 7.68, and 9.60 g per plant), P
2
O
5
(0, 0.98, 1.47, 2.44, and 3.42 g per plant), and
K
2
O (0, 2.30, 4.61, 6.91, and 9.22 g per plant) [
72
]. Following the methodology proposed
Horticulturae 2021,7, 426 10 of 13
by [
25
], sap NO
3−
-N, NH
4+
, PO
4
H
3
, and K were evaluated. Sap analysis was proven
to indicate the crop N status, as it responded linearly to the increasing fertilization rates.
Another interesting finding was the negative correlation (
−
0.88) between applied P and
sap NO
3−
-N, as increasing P rates resulted in reduced sap NO
3−
-N levels. Leaf analysis
was more effective than sap analysis to show the current P and K status. However, the
sap P and K values could be a function of the crop growth stage, as mentioned previously
by other authors [
34
]. Nevertheless, sap analysis had a higher sensitivity for determin-
ing interactions and antagonisms among nutrients; therefore, it seems to be an effective
complementary tool for assessing grapevine nutrient status.
Table 5.
Sap nutrient concentration levels for ‘Red Globe’ grapevine (Vitis vinifera) during the crop
cycle. Adapted from [26].
Crop Stage Sap Nutrient Concentration (mg L−1)
NO3−-N P K+Ca2+ Mg2+
Vegetative flush 1700 155 2800 600 480
Blooming 300 530 2000 1200 1000
Veraison 550 870 3350 1400 1400
5. Conclusions
As agriculture moves towards precision, sap analysis is a complementary tool for
nutrition management in citrus production. Limitations regarding methodologies and
results interpretation are gaps that might be filled with appropriate research. Much work
is still to be conducted regarding methodology standardization and the determination of
reference levels in HLB-affected and non-affected trees. If managed appropriately, sap
analysis can optimize fertilizer application to meet tree nutrient requirements, reduce envi-
ronmental impacts, and improve sustainability. Before the scientific community determines
a standardized methodology and reliable sufficiency ranges, sap analysis should be used
with caution.
Author Contributions:
Conceptualization, E.E., G.L., N.A.B. and R.S.F.; methodology, E.E., G.L.,
N.A.B. and R.S.F.; investigation, E.E., G.L., N.A.B. and R.S.F.; resources, R.S.F.; writing—original draft
preparation, E.E., G.L., N.A.B. and R.S.F.; writing—review and editing, E.E. and R.S.F.; supervision,
R.S.F.; project administration, R.S.F.; funding acquisition, R.S.F. All authors have read and agreed to
the published version of the manuscript.
Funding:
This research was partially funded by the Southern SARE On-Farm Research Grant Award
#2020-38640-31521.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments:
We thank W. Cody Estes Sr. (Estes Groves, Inc.) and Scott D. Wall (New Age
Laboratories) for technical support. This review was written to complement the information provided
by the 2021 Plant Sap Analysis Workshop in Citrus Production organized by R.S.F. and E.E. available
at https://irrec.ifas.ufl.edu/faculty-members-/ferrarezi/plant-sap-analysis.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the
interpretation of data, in the writing of the manuscript, or in the decision to publish the results.
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