Available via license: CC BY
Content may be subject to copyright.
applied
sciences
Article
Characterization of Nutrient Disorders
of Cannabis sativa
Paul Cockson 1, * , Hunter Landis 1,2, Turner Smith 1, Kristin Hicks 2and Brian E. Whipker 1
1Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695, USA;
hlandis@g.clemson.edu (H.L.); jtrexsmith@gmail.com (T.S.); bwhipker@ncsu.edu (B.E.W.)
2North Carolina Department of Agriculture and Consumer Services, Raleigh, NC 14142, USA;
Kristin.Hicks@ncagr.gov
*Correspondence: pncockso@ncsu.edu
Received: 23 September 2019; Accepted: 9 October 2019; Published: 18 October 2019
Abstract:
Essential plant nutrients are needed at crop-specific concentrations to obtain optimum
growth or yield. Plant tissue (foliar) analysis is the standard method for measuring those levels in
crops. Symptoms of nutrient deficiency occur when those tissue concentrations fall to a level where
growth or yield is negatively impacted and can serve as a visual diagnostic tool for growers and
researchers. Both nutrient deficiency symptoms and their corresponding plant tissue concentrations
have not been established for cannabis. To establish nutrient concentrations when deficiency or toxicity
symptoms are expressed, Cannabis sativa ‘T1’ plants were grown in silica sand culture, and control
plants received a complete modified Hoagland’s all-nitrate solution, whereas nutrient-deficient
treatments were induced with a complete nutrient formula withholding a single nutrient. Toxicity
treatments were induced by increasing the element tenfold higher than the complete nutrient formula.
Plants were monitored daily and, once symptoms manifested, plant tissue analysis of all essential
elements was performed by most recent mature leaf (MRML) tissue analysis, and descriptions and
photographs of nutrient disorder symptomology were taken. Symptoms and progressions were
tracked through initial, intermediate, and advanced stages. Information in this study can be used to
diagnose nutrient disorders in Cannabis sativa.
Keywords:
macronutrients; micronutrients; cannabis; deficiency; toxicity; fertility; symptomology;
hemp; diagnostics; plant tissue analysis; CBD; THC; foliar
1. Introduction
Due to recent changes in legislation both at the federal and state levels, there has been a surge of
interest in the growing, processing, selling, and using of products containing cannabidiol (CBD), derived
from hemp flowers. Hemp is legally defined as Cannabis sativa strains with a tetrahydrocannabinol
(THC) concentration no greater than 0.3% in any part of the plant (Congress, [
1
,
2
]). Cannabis sativa
strains with a THC concentration greater than 0.3% in any part of the plant are considered marijuana.
Cannabis sativa contains over 100 cannabinoids, which include THC and CBD. It is well known that
THC has psychoactive effects. Many have reported health benefits from marijuana, which may be
associated with non-THC cannabinoids, such as CBD. The broad interest in CBD is for health benefits
similar to marijuana but without the psychoactive effects of THC.
Hemp has historically been grown for fiber and seed, and due to recent changes in legislation, it is
being grown for flowers. Hemp grown for flowers (floral hemp) follows a horticultural production
model either in a greenhouse or bedded field compared to fiber and seed hemp, which follows an
agronomic production model.
Appl. Sci. 2019,9, 4432; doi:10.3390/app9204432 www.mdpi.com/journal/applsci
Appl. Sci. 2019,9, 4432 2 of 14
There is little published research investigating fertility requirements for floral hemp. As applied
research is conducted to determine nutrient rates to maximize yield and minimize inputs, as well as to
develop a target range of plant sufficiency ranges to aid in nutrient management, this study provides
an invaluable basis to identify nutrient deficiency in the field and to develop sufficiency ranges where
nutrient corrective action can be made before visual symptoms are expressed.
The impacts of plant nutrition on plant growth and yield, as well as plant primary and secondary
metabolites, are well-established (Il’in, [
3
]). In hemp fiber varieties, Bosca et al. [
4
] reported higher
levels of nitrogen increased plant leaf weight and decreased leaf THC content, presumably due to
THC dilution. In a marijuana strain, phosphorus treatments had greater combined leaf and flower dry
weight, as well as higher THC concentration, compared to the no phosphorus treatment (Coffman
and Gentner, [
5
,
6
]). Hemp producers are seeking THC levels <0.3% and high CBD concentrations (i.e.,
10–20%). Given the high energy and resource requirements for plants to produce secondary plant
metabolites, such as cannabinoids (Taura et al., [
7
,
8
]), it would be reasonable to assume that nutritive
disorders would impact the production and quality of these metabolites.
Plant tissue analysis has been used extensively for many decades to evaluate the nutritional
status of a crop. Nutrient sufficiency levels (the tissue concentration at which growth or yield is
not limited) have been established for most major agricultural and horticultural plants. Because
cannabis has not been widely grown legally, nutrient sufficiency ranges have not been established.
The development of sufficiency ranges for the essential plant nutrients would require extensive rate
studies that measure both elemental concentrations in the leaf and the yield parameters of both
floral biomass and cannabinoid concentrations. Where sufficiency ranges of a crop are not available,
survey ranges can be used to approximate the nutritional status of the plant. Survey ranges for
Cannabis sativa in greenhouse nursery production have been published by Bryson et al. [
9
], and more
recently, a survey of five hemp cultivars in greenhouse production, including the cultivar used in this
study, by Landis et al. [10]. These tissue values are useful for cannabis growers as they aid in fertility
management. This study adds to this body of knowledge as the complete fertilizer controls can serve
as an additional set of survey ranges for cannabis.
The second contribution of this study results in information about nutrient deficiency levels
in the leaf tissue of cannabis. Once a plant begins showing visual symptoms of impaired growth,
there is a reduction in plant health or yield is implicit. In this study, when plants began showing
deficiency symptoms for each nutrient, most recently, mature leaf samples were analyzed for that
nutrient. This information can be used by growers and researchers to confirm the visual diagnosis
with leaf concentrations.
Additionally, no visual guides of nutrient deficiencies in cannabis supported with leaf tissue
analysis and documenting a progression of symptomology have been published. Tracking the specific
symptomologies of various nutritional disorders over time is important because symptomologies
change in appearance and location as the deficiency progresses, making correct diagnosis challenging.
Therefore, this study was conducted to provide cannabis growers and researchers with descriptions
of nutrient disorders, high-quality images to track the progression of these disorders, and leaf tissue
nutrient concentrations associated with documented deficiency symptomology.
2. Materials and Methods
Cuttings were taken from a hemp Cannabis sativa ‘T1’on 3 July 2018 (Ryes Greenhouses: Sanford,
NC, USA) and stuck into 72-cell plug trays filled with a substrate mix of 80:20 (v:v) Canadian sphagnum
peat moss (Conrad Fafard, Agawam, MA, USA) and horticultural coarse perlite (Perlite Vermiculite
Packaging Industries, Inc., North Bloomfield, OH, USA) amended with dolomitic lime at 8.875 kg/m
3
(Rockydale Agricultural, Roanoke, VA, USA) and wetting agent (Aquatrols, Cherry Hill, NJ, USA) at
600.3 g/m
3
. After three weeks of rooting, plugs were transplanted (27 July 2018) into 15.24-cm diameter
(1.76 L) plastic pots filled with acid-washed silica-sand (Millersville #2 (0.8 to 1.2 mm diameter) from
Southern Products and Silica Co., Hoffman, NC, USA).
Appl. Sci. 2019,9, 4432 3 of 14
The experiment was conducted in a glass greenhouse in Raleigh, NC, USA (35
◦
N latitude),
with 24◦C/20◦C
(D/N) temperature setpoints. Plugs were transplanted, and nutrient treatments started
immediately in the automated, recirculating irrigation system made from 10.2-cm diameter PVC pipe
(Charlotte Plastics, Charlotte, NC, USA), fit with 12.7-cm diameter openings to hold the pots. Control
plants were grown with a complete modified Hoagland’s all-nitrate solution consisting of 15 mM
nitrate-nitrogen (NO
3−
), 1 mM phosphate-phosphorus (H
2
PO
4−
), 6 mM potassium (K
+
), 5 mM calcium
(Ca
2+
), 2 mM magnesium (Mg
2+
), and 2 mM sulfate-sulfur (SO
42−
) (Hoagland and Arnon, [
11
]); plus
72
µ
M iron (Fe
2+
), 18
µ
M manganese (Mn
2+
), 3
µ
M copper (Cu
2+
), 3
µ
M zinc (Zn
2+
), 45
µ
M boron
(BO
33−
), and 0.1
µ
M molybdenum (MoO
42−
). Nutrient deficiencies began at transplant and were
induced by withholding a single nutrient from this solution. Boron (B) and manganese (Mn) toxicities
were induced by increasing the concentration tenfold higher than the complete nutrient formula.
Reagent grade chemicals and deionized (DI) water (18 M
Ω
) were used to formulate treatment solutions.
The plants were drip-irrigated with a sump-pump (model 1A, Little Giant Pump Co., Oklahoma City,
OK, USA) system as needed between 6:00 and 18:00 hours. Irrigation solution drained from the pot
and was captured for reuse. Nutrient solutions were replaced weekly. The experiment was terminated
9 weeks after treatments began.
Plants were tracked daily, and deficiency symptoms were photographed at the initial, intermediate,
and advanced stages of symptomology. Plant anatomy terminology used to describe deficiency
symptoms is given in Figure 1. Upon initial symptom development, four plants were selected for
sampling. The remaining treatments, which were not symptomatic, were grown until visual symptoms
appeared and then were harvested. At the onset of initial visual symptomology, whole plants (n=4)
were destructively harvested, and most recent mature leaves (MRML) were subsampled and rinsed
with DI water, washed in a solution of 0.5 M HCl, and again rinsed with DI water. Leaf tissue
(MRML) was taken below the meristematic stem regions (apical meristem, axillary meristems, tertiary
meristematic regions, etc.), and only recently matured leaves were sampled. Leaf maturity and
morphology were determined based on observational leaf maturity and from indices hybridized from
Heslop–Harrison and Heslop–Harrison [
12
] and Mediavilla et al. [
13
] and from established standard
leaf tissue harvesting protocols (Bryson et al., [
9
]). The remaining plant material after MRML was
harvested and was placed in a separate container for aerial tissue biomass determination. Leaf tissues
and their respective remaining biomass were dried at 70
◦
C for 24 hours. Dried leaf tissue was ground
in a mill (Thomas Wiley
®
Mini-Mill; Thomas Scientific, Swedesboro, NJ, USA) with a 20-mesh (1 mm)
screen and analyzed for nutrient concentrations by the North Carolina Department of Agriculture
and Consumer Services (NCDA&CS) Agronomic Division (NCDA&CS, [
14
]). Total plant dry weight
(DW) was calculated by adding the oven-dry weight of the leaf tissue to the oven-dry weight of the
remaining plant biomass (Table 1). Details about experimental setup, fertilizers, and design can be
found in Barnes et al. [
15
]. Data were analyzed with SAS version 9.4 (SAS Institute, Cary, NC, USA) and
subjected to analysis of variance (ANOVA) using PROC ANOVA and (GLM) PROC GLM. Where F-tests
indicated evidence of significant differences among means, LSD (Least Significant Difference)
(P≤0.05
)
was used.
Appl. Sci. 2019,9, 4432 4 of 14
Figure 1. Sketch of a branch of Cannabis sativa ‘T1’ plant showing plant anatomy and terminology.
Table 1.
Mean dry weights of Cannabis sativa ‘T1’ plants grown with a deficient macronutrient treatment
compared to plants grown with a complete fertilizer.
Dry Weight (g)
Treatment –N †–P –K –Ca –S –Mg
Control 13.46 20.00 44.20 20.00 20.00 20.00
Disorder 6.77 15.44 32.27 16.63 18.01 18.01
*** ‡NS ** NS NS NS
†
Nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), sulfur (S), and magnesium (Mg).
‡
*, **, or *** indicate
statistically significant differences between sample means (n=4) based on F-tests at P<0.05, P<0.01, or P<0.001,
respectively. NS (not significant) indicates the F-test difference between sample means was P>0.05.
3. Results
3.1. Macronutrient Disorders
3.1.1. Nitrogen
Symptoms of nitrogen (N) deficiency developed 6 weeks after treatment. The plants first displayed
slight stunting when compared to the control. Initially, the plants developed a slight overall yellowing
or paling of the lower foliage (Figure 2). As the deficiency progressed, the yellowing became more
intense and progressed up from the bottom-most leaves to the middle foliage. In advanced symptoms,
the yellowing leaves became completely yellow and eventually turned necrotic and abscised.
Figure 2.
Nutritional disorders of nitrogen (N), phosphorus (P), and calcium (Ca) deficiency in
Cannabis sativa ‘T1’ plants. These pictures display the symptomological progression of nutritional
disorders from initial, intermediate, through advanced.
Appl. Sci. 2019,9, 4432 5 of 14
Foliar N concentrations were 62% lower in the N deficient plants than in the controls. N deficient
plants contained 1.62% N, while the control plants contained 4.28% N (Table 2). Foliar N concentrations
in control plants were within the published greenhouse survey range of 3.30–4.76% N (Bryson et al. [
9
]).
Plants grown in N deficient conditions produced 50% less biomass when compared to the control
(Table 1).
Table 2.
Foliar nutrient concentrations of Cannabis sativa ‘T1’ grown with a deficient macronutrient
compared to plants grown with a complete fertilizer regime.
Treatment –N†–P –K –Ca –Mg –S
Tissue nutrient concentration (% dry weight)
Element N P K Ca Mg S
Complete 4.28 0.43 2.85 3.73 0.61 0.41
Disorder 1.62 0.09 0.41 0.39 0.12 0.11
*** ‡*** *** *** *** ***
Survey
Range 13.30–4.76 0.24–0.49 1.83–2.35 1.47–4.42 0.4–0.81 0.17–0.26
†
Nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S),
‡
*, **, or *** indicate
statistically significant differences between sample means (n=4) based on F-tests at P<0.05, P<0.01, or P<0.001,
respectively. NS (not significant) indicates the F-test difference between sample means was P>0.05.
1
Reference
survey values from Bryson et al. (2014).
3.1.2. Phosphorus
Symptoms of phosphorus (P) deficiency developed 7 weeks after treatment. The plants first
displayed slight stunting when compared to the control. Initially, the plants developed olive-green
spots in an irregular spotting pattern along with the leaflets on the lower and older leaves (Figure 2).
As symptoms progressed, the olive-green spots developed into larger olive-green spots that appeared
sunken and almost wet in appearance with some marginal necrosis. In advanced symptoms,
the yellowing leaves became severely olive spotted with large areas showing symptoms, and in
severe cases, large necrotic portions developed.
Foliar P concentrations were 79% lower in the P deficient plants than in the controls. Treated
deficient plants contained 0.09% P, while the control plants contained 0.43% P (Table 2). Foliar P
concentrations in control plants were within the published survey range of 0.24–0.49% (Bryson et al.,
2014). Plants grown in P deficient conditions did not result in statistically significant differences in
biomass when compared to the control (Table 1).
3.1.3. Calcium
Symptoms of Ca deficiency developed five weeks after treatment. The plants first displayed slight
stunting when compared to the control. The growing tips and newly expanding leaves showed signs
of stunting and irregular growth habits (Figure 2). As the newly expanding leaves developed, the basal
portion of the leaflets remained narrower and displayed a lighter green coloration when compared to
the leaflet tip (Figure 2). As symptoms progressed, the yellowing at the leaflet basal portion became
more intense, and the leaflets began to show symptoms of interveinal chlorosis. The new leaves that
expanded showed severe stunting and marginal necrosis, resulting in leaves with irregular geometries
and orientations (Figure 2). In advanced symptoms, the yellowing leaves and growing tips became
necrotic. The death of the growing tip caused a proliferation of axillary shoot development to occur,
resulting in a more branched architecture (Figure 2).
Foliar Ca concentrations were 90% lower in the Ca-deficient plants than in the controls. Ca-deficient
plants contained 0.39% Ca, while the control plants contained 3.73% Ca (Table 2). Foliar Ca
concentrations in control plants were within the published survey range of 1.47–4.42%
(Bryson et al., [9])
.
Appl. Sci. 2019,9, 4432 6 of 14
Plants grown in Ca-deficient conditions did not result in statistically significant differences in biomass
when compared to the control (Table 1) when sampled at the onset of visual symptoms.
3.1.4. Sulfur
Symptoms of sulfur (S) deficiency developed 7 weeks after treatment. The plants first displayed
a slight overall yellowing of the foliage, especially in the middle of the plant. The yellowing leaves
had a more pronounced yellowing at the base of the leaflets (Figure 3). As symptoms progressed,
the yellowing at the leaflet basal portion became more intense, and the yellowing intensified on newly
expanding leaves (Figure 3). In advanced symptoms, the leaves became a very pale yellow in coloration,
especially around the midrib and base of the leaflets (Figure 3).
Figure 3.
Nutritional disorders of sulfur (S), magnesium (Mg), and potassium (K) deficiency in
Cannabis sativa ‘T1’ plants. These pictures display the symptomological progression of nutritional
orders as they progress from initial, intermediate, and advanced.
Foliar S concentrations were 73% lower in the S-deficient plants than in the controls. S-deficient
plants contained 0.11% S, while the control plants contained 0.41% S (Table 2). Foliar S concentrations
in control plants were above the published survey range of 0.17–0.26% (Bryson et al., [
9
]). Plants grown
in S-deficient conditions did not result in statistically significant differences in biomass when compared
to the control (Table 1).
3.1.5. Magnesium
Symptoms of Mg deficiency developed 7 weeks after treatment. Initial symptoms developed with
slight yellowing of the interveinal regions of the lower and older foliage (Figure 3). As symptoms
progressed, the interveinal yellowing became more pronounced and intensified on the older leaves
(Figure 3). In advanced symptoms, the leaves showed a very stark contrast between the green veins
and the yellow interveinal regions, with some regions becoming necrotic (Figure 3).
Foliar Mg concentrations were 80% lower in the Mg-deficient plants than in the controls.
Mg-deficient plants contained 0.12% Mg, while the control plants contained 0.61% (Table 2). Foliar Mg
concentrations in control plants were within the published survey range of 0.40–0.81%
(Bryson et al., [9])
.
Plants grown in Mg-deficient conditions did not result in statistically significant differences in biomass
when compared to the control (Table 1).
Appl. Sci. 2019,9, 4432 7 of 14
3.1.6. Potassium
Symptoms of K deficiency developed 9 weeks after treatment. Initial symptoms developed as a
yellowing of the leaf margin, especially the saw-tooth of the leaflets, on the lower and older foliage
(Figure 3). As symptoms progressed, the marginal yellowing became more pronounced and intensified
on the older leaves, expanding inward toward the midrib (Figure 3). In advanced symptoms, the leaflet
margins yellowed, and some regions of tan necrosis developed, especially along the saw-tooth margin
of the leaflets (Figure 3).
Foliar K concentrations were 86% lower in the K-deficient plants than in the controls. K-deficient
plants contained 0.41% K, while the control plants contained 2.85% K (Table 2). Foliar K concentrations
in control plants were above the published survey range of 1.83–2.35% (Bryson et al., [
9
]). Plants grown
in K-deficient conditions weighed 27% less when compared to the control (Table 1).
3.2. Micronutrient Disorders
3.2.1. Manganese
Symptoms of Mn deficiency developed 6 weeks after treatment. Initially, the plants developed a
bright yellow netted interveinal chlorosis on the upper and central foliage (Figure 4). This chlorotic
netting initiated at the midrib of the leaflets and spread outward toward the leaf margin as symptoms
progressed. In advanced symptoms, the interveinal netting became very distinct against the green
veinal regions. Additionally, the interveinal regions developed small tan necrotic regions on the leaf
surface (Figure 4).
Figure 4.
Nutritional disorders of manganese (Mn), boron (B), and copper (Cu) deficiency in
Cannabis sativa ‘T1’ plants. These pictures display the symptomological progression of nutritional
orders as they progress from initial, intermediate, and advanced.
There were no statistically significant differences in the dry weights of the control plant and the
Mn-deficient plants (Table 3). Despite the lack of statistical significance, foliar Mn concentrations
were 74% lower in the Mn-deficient plants than in the controls. Deficiency treated plants contained
7.56 mg
·
kg
−1
Mn, while the control plants contained 29.40 mg
·
kg
−1
(Table 4). Foliar Mn concentrations
in the control plants were slightly below the published sufficiency range of 41–93 mg
·
kg
−1
Mn
(Bryson et al., [9]).
Appl. Sci. 2019,9, 4432 8 of 14
Table 3.
Mean dry weights of Cannabis sativa ‘T1’ plants grown with a deficient or toxic micronutrient
treatment compared to plants grown with a complete fertilizer regime.
Dry Weight (g)
Treatment –B †+B –Cu –Fe –Mn +Mn –Mo –Zn
Control 13.46 19.99 44.20 44.20 13.46 19.99 44.20 44.20
Disorder 9.69 21.16 24.23 43.42 13.09 18.70 39.91 43.23
*‡NS ** NS NS NS NS NS
†
Boron (B), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), and zinc (Zn).
‡
*, **, or *** indicate
statistically significant differences between sample means (n =4) based on F-tests at P<0.05, P<0.01, or P<0.001,
respectively. NS (not significant) indicates the F-test difference between sample means was P>0.05.
Table 4.
Foliar nutrient concentrations of Cannabis sativa ‘T1’ grown with a deficient micronutrient
treatment compared to plants grown with a complete fertilizer regime.
Treatment –B †+B –Cu –Fe –Mn +Mn –Mo –Zn
Tissue nutrient concentration (mg·kg−1)
Element B B Cu Fe Mn Mn Mo Zn
Complete
58.58 64.60 4.65 111.75 29.40 31.13 1.46 25.33
Disorder 2.46 671.75 1.41 60.1 7.56 47.88 0.06 10.7
*** ‡*** *** *** *** *** *** ***
Survey
Range 156–105 56–105 5.0–7.1 100–150 41–93 41–93 0.5–1.5 24–52
†
Boron (B), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), and zinc (Zn).
‡
*, **, or *** indicate
statistically significant differences between sample means (n =4) based on F-tests at P<0.05, P<0.01, or P<0.001,
respectively. NS (not significant) indicates the F-test difference between sample means was P>0.05.
1
Reference
survey values from Bryson et al. (2014).
3.2.2. Manganese Toxicity
Symptoms of Mn toxicity developed 7 weeks after treatment. Initially, the plants developed a
marginal yellowing of the lower leaves (Figure 5). This yellowing intensified and moved inward on
the leaf surface toward the midrib. In advanced symptoms, the leaf margin became necrotic, and the
leaf appeared severely chlorotic. In some cases, the symptomatic leaves abscised (Figure 5).
Figure 5.
Nutritional disorders of manganese toxicity (Mn) and boron toxicity (B) in Cannabis sativa ‘T1’
plants. These pictures display the symptomological progression of nutritional orders as they progress
from initial, intermediate, and advanced.
Appl. Sci. 2019,9, 4432 9 of 14
Foliar Mn concentrations were 53% higher in the Mn toxicity treatment plants than in the controls.
Toxicity treated plants contained 47.88 mg
·
kg
−1
Mn, while the control plants contained 31.13 mg
·
kg
−1
(Table 4). Foliar Mn concentrations, in both toxicity treated and control plants, were within the
published survey range of 41–93 mg
·
kg
−1
(Bryson et al., [
9
]). While the toxicity treatments were
higher, they were still within the accepted ranges for most crops. There were no statistically significant
differences in the dry weights of the control plant and the Mn toxicity plants (Table 3). Higher
concentrations of Mn will need to be used to further refine the threshold for toxicity symptomology.
3.2.3. Boron
Symptoms of B deficiency developed 6 weeks after treatment. Initially, the plants developed slight
stunting in their growth habits when compared to the control. Upon closer inspection, the stunting
proliferated from the growing tips of the B-deficient plants. The growing tips and newer foliage
displayed a distorted growth pattern. The new and expanding leaflets were smaller and narrower at
the leaflet base when compared to the tip (Figure 4). In advanced symptoms, the new and expanding
leaves displayed severe distortion. The leaflet margins became necrotic, and the leaves distorted
severely, curling inward and down as well as at odd angles from the petiole (Figure 4). In the most
advanced stages, the growing tips died and became necrotic, and the whole plant showed severe
wilting due to the death of the root tips and subsequent loss of root biomass.
Foliar B concentrations were 96% lower in the B-deficient plants than in the controls. Deficiency
treated plants contained 2.46 mg
·
kg
−1
B, while the control plants contained 58.58 mg
·
kg
−1
(Table 4).
Foliar B concentrations in control plants were within the published survey range of 56–105 mg
·
kg
−1
B (Bryson et al., [
9
]). Plants grown in B-deficient conditions produced 28% less biomass when
compared to the control (Table 3). This is most likely due to the death of the apical growing tip due to
B-deficient conditions.
3.2.4. Boron Toxicity
Symptoms of B toxicity appeared 7 weeks after treatment. Initially, the plants developed a
marginal yellowing of the lower leaves (Figure 5). This yellowing intensified along the leaf margin
and moved inward toward the midrib of the leaflets. In advanced symptoms, the leaf margin turned
brown and eventually became necrotic (Figure 5).
Foliar B concentrations were >10 fold higher in the B toxicity plants than in the controls. Toxicity
treated plants contained 671.75 mg
·
kg
−1
B, while the control plants contained 64.60 mg
·
kg
−1
(Table 4).
Foliar B concentrations in control plants were within the published survey range of 56–105 mg
·
kg
−1
B
(Bryson et al., [
9
]). There were no statistically significant differences in the dry weights of the control
plant and the B toxicity plants (Table 3).
3.2.5. Copper
Copper deficiency manifested very late in the growth of the plants, only displaying symptomology
after 9 weeks. Initially, the plants developed slight stunting in their growth habits when compared to
the control. This stunting was accompanied by a slight distortion of the newer and expanding leaves,
especially at the leaflet base. The base of the leaflets was narrower and displayed slight yellowing
(Figure 5). In advanced symptoms, the new and expanding leaves displayed a more pronounced basal
narrowing and distortion and started to exhibit marginal interveinal chlorosis (Figure 5). In the most
advanced stages, the whole leaf displayed a fine interveinal chlorosis, and the leaf margin distorted,
slightly curling in and downward. The new and expanding leaves also displayed a slight decrease in
turgidity (Figure 5).
Foliar Cu concentrations were 70% lower in plants subjected to Cu-deficient conditions. Deficiency
treated plants contained 1.41 mg
·
kg
−1
Cu, while the control plants contained 4.65 mg
·
kg
−1
(Table 4).
Foliar Cu concentrations in control plants were slightly below the published survey range of
Appl. Sci. 2019,9, 4432 10 of 14
5–7.1 mg
·
kg
−1
Cu (Bryson et al., [
9
]). Plants grown in Cu-deficient conditions produced 45% less
biomass when compared to the control (Table 3).
3.2.6. Iron
Symptoms of upper leaf interveinal chlorosis developed on Fe-deficient plants after 9 weeks
of iron deficiency treatments. The leaves were lighter in appearance compared to control plants,
and symptoms spread throughout the upper half of the foliage, especially around the growing tip and
newly expanding leaves (Figure 6). Symptoms of Fe stress began as a slight marginal yellowing of the
leaflets, especially around the base of the leaf (Figure 6). As symptomology progressed, the upper
foliage displayed interveinal chlorosis on the new and expanding leaves, while the lower and mid
foliage displayed a healthy dark green coloration (Figure 6).
Figure 6.
Nutritional disorders of iron (Fe) and zinc (Zn) deficiencies in Cannabis sativa ‘T1’ plants.
These pictures display the symptomological progression of nutritional orders as they progress from
initial, intermediate, and advanced.
Foliar Fe concentrations were 46% lower in plants subjected to Fe-deficient conditions when
compared to the controls. Deficiency treated plants contained 60.08 mg
·
kg
−1
Fe, while the control
plants contained 111.75 mg
·
kg
−1
(Table 4). Foliar Fe concentrations in control plants were within the
published survey range of 100–150 mg
·
kg
−1
(Bryson et al., [
9
]). Plants grown in Fe-deficient conditions
produced a similar amount of biomass when compared to the control (Table 3).
3.2.7. Molybdenum
Visual symptoms did not develop on Mo-deficient plants after 9 weeks of deficiency treatments.
Despite a lack of visual symptoms, foliar Mo concentrations were 96% lower in plants subjected
to Mo-deficient conditions when compared to the controls. Deficiency treated plants contained
0.06 mg
·
kg
−1
Mo, while the control plants contained 1.46 mg
·
kg
−1
(Table 4). Foliar Mo concentrations
in control plants were within the published survey range of 0.5–1.5 mg
·
kg
−1
Mo (
Bryson et al., [9])
.
Despite the statistical significance of the tissue concentrations, there was no statistical difference in dry
weights of the Mo deficient plants when compared to the controls (Table 3).
Appl. Sci. 2019,9, 4432 11 of 14
3.2.8. Zinc
Zinc deficiency manifested very late in the growth of the plants, only displaying symptomology
after 9 weeks. Deficiency symptoms manifested first as a marginal yellowing on the newer foliage and
expanding leaves (Figure 6). This yellowing was concentrated mostly in the margin of the leaf and
along the toothed portions of the leaflets. As symptoms progressed, these yellow marginal regions
developed into tan irregularly shaped necrotic regions along the leaf margin (Figure 6).
Foliar Zn concentrations were 58% lower in plants subjected to Zn-deficient conditions when
compared to the controls. Deficiency treated plants contained 10.70 mg
·
kg
−1
, while the control plants
contained 25.33 mg
·
kg
−1
(Table 4). Foliar Zn concentrations in control plants were within the published
survey range of 24–52 mg
·
kg
−1
Zn (Bryson et al., [
9
]). Plants grown in Zn-deficient conditions produced
similar amounts of biomass when compared to the control (Table 3). Despite the statistical significance
of the Zn tissue concentrations (Table 4), there was no statistical difference in dry weights between the
Zn-deficient plants and the control plants (Table 3).
4. Discussion
While some anecdotal deficiency symptoms for cannabis are available in lay publications,
scientifically rigorous symptomology, particularly symptomological progression, is very limited or
non-existent in the literature. In this study, most documented symptoms of deficiencies were consistent
with descriptions from current literature for other plant species (Bryson et al., [
9
]; Barnes, [
16
]; Barker
and Pilbeam, [
17
]; Gibson et al., [
18
]) with some exceptions. Concentrations of N, P, K, Ca, Mg, and S
in the leaves when deficiency symptomology first appeared were 1.62, 0.09, 0.41, 0.39, 0.12, and 0.11%,
respectively. Concentrations of B, Cu, Fe, Mn, Mo, and Zn in the leaves when deficiency symptomology
first appeared were 2.46, 1.41, 60.1, 7.56, 0.06, and 10.7 mg·kg−1, respectively.
Plants grown without Mo did not exhibit leaf symptomology nor less dry matter production
despite leaf tissue values being 96% lower than the complete controls. While visual symptoms of
Cu deficiency displayed the interveinal chlorosis in younger leaves documented in other plants,
the Cu-deficient cannabis also showed an odd wilting pattern (Figure 4). This wilting in Cu-deficient
hemp plants is mentioned in fiber hemp (Van der Werf, [
19
]). Copper is important in cell wall
metabolism (Yruela, [
20
]). The condition which Van der Werf [
19
] referred to as “’gummi’-hemp” may
indicate that Cu is needed in greater quantities in hemp than in other species, especially when grown
for fiber. While these symptoms of wilting and lodging were in fiber hemp, which has a very vigorous
vertical growth habit, the same process could be occurring in floral hemp cultivars.
Additionally, B-deficient treated plants displayed similar wilting tendencies. Upon further
inspection of the roots, it was shown that the terminal growing tip had died and turned necrotic,
and there were many axillary roots due to the loss of apical dominance. Boron is important in cell wall
development and elongation of plant cell walls, especially in the radicle and other root meristematic
regions (Hu and Brown, [
21
]; Whittington, [
22
]). The wilting seen could have been due to a lack of root
mass due to an underdeveloped root system. Given all plants were irrigated for the same amount of
time, it is feasible to assume that the wilting was due to a lack of water uptake because root growth
was impaired.
Nutrients varied in how rapidly deficiency symptoms were visually apparent. In treatments
where N, P, Ca, Mg, S, Mn, and B were withheld, deficiency symptoms were observed within 6 to
7 weeks. Conversely, where K, Zn, Cu, Fe were withheld, visual deficiencies only occurred after
9 weeks and, as previously noted, not at all in Mo. The use of visual symptomology as a diagnostic tool
may be more useful for the nutrients that demonstrate symptoms sooner rather than later. However,
for field and greenhouse floral hemp production, visual symptoms may express early enough in the
season where rescue nutrient applications can be made. Additionally, routine MRML tissue sampling
during the season will identify if nutrients are getting near the concentrations that show symptoms,
thus allowing a correcting nutrient application before symptoms are expressed.
Appl. Sci. 2019,9, 4432 12 of 14
Some nutrient deficiencies had a significant suppressive effect on yield, as measured by whole
plant dry weight, while withholding other nutrients did not affect total biomass. Deficiencies of N, K,
B, and Cu produced significantly less crop biomass by 50, 27, 28, and 45%, respectively, as compared
to the control. While all other nutrient deficiencies had less biomass, none suppressed yield at a
significance level of P>0.05. Above-ground biomass may not be the appropriate metric for testing
critical levels in these nutrients, and other factors, such as secondary metabolite production, maybe a
better indicator of the minimum nutrient concentrations for optimum production.
The development of true cannabis sufficiency ranges for each essential element requires individual
rate studies coupled with measurements of both dry matter production and cannabinoid yield. Until
this extensive work can be completed, survey ranges are useful tools for estimating adequate nutrient
levels. In this study, the control samples served as survey ranges, which can be compared to other
published survey ranges for cannabis (Bryson et al., [
9
]; Landis et al., [
10
]). Some elements in the
control samples were above (K and S) or below (Cu and Mn) the reported values from
Bryson et al. [9].
Other control values found in this study were within or above the listed values for Cannabis sativa
‘T1’ except for Mn values, which were below the listed values from Landis et al. [
10
]. While the
ranges reported by Bryson et al. [
9
] are useful guides for estimating healthy nutrient levels in hemp
tissue, it is important to remember that they are the survey ranges from plants grown in a production
nursery of an unknown replicate number and unknown hemp cultivar and may not accurately predict
optimum nutrient levels in all production systems in all cultivars. Recently,
Landis et al. [10] found
that significant differences in nutrient concentrations occurred among CBD cultivars, suggesting that
broader target nutrient ranges may be appropriate for cannabis.
Boron toxicity was first observed at an accumulated foliar leaf tissue concentration of
671.75 mg
·
kg
−1
, and the symptoms (marginal chlorosis and necrosis in the older leaves) were consistent
with B toxicity in other plants. Plants vary in their sensitivity to boron. Toxicity symptoms occur in
strawberry at foliar concentrations as low as 120 mg
·
kg
−1
(Haydon, [
23
]), suggesting that cannabis
may be more tolerant of excess B than some crops.
5. Conclusions
This work serves as a baseline for nutritive values in the Cannabis sativa ‘T1’ cultivar, and it is
recommended that the upper and lower ranges from this study, Landis et al. [
10
],
and Bryson et al. [9] be
used when evaluating leaf tissue nutrient concentrations. The nutrient disorders described in this
study provide hemp growers and researchers with detailed descriptions and high-quality diagnostic
images to better identify nutrient disorders. Additionally, previous works were lacking in diagnostic
rigor and did not report foliar nutrient values. These nutrient values can also help growers to monitor
their crops and can be used to make improved fertilization decisions. With the exception of N,
K, B, and Cu, most disorders had no significant effect on overall plant dry weight at the onset of
symptoms. However, if measurements of plant dry weight had also been collected at intermediate and
advanced stages of symptomology, greater negative impacts on growth would likely have occurred.
In addition, measurements of dry weights of floral parts at harvest and concentrations of cannabinoids
from those floral could offer insight into the effects of these essential nutrients on plant growth and
yield parameters other than vegetative biomass. These data illustrate the importance of recognizing
nutritional disorders at an early stage to implement corrective procedures in order to optimize yield
and produce a successful crop.
Author Contributions:
Conceptualization: B.E.W. and P.C.; Methodology: B.E.W., P.C., H.L., and T.S.; Software:
B.E.W., P.C., H.L., and K.H.; Validation: P.C., K.H. and H.L.; Formal analysis: P.C., H.L., and K.H.; Investigation:
P.C., B.E.W., H.L. and T.S.; Resources: P.C. and B.E.W.; Data curation: P.C.; Writing—original draft preparation:
P.C., B.E.W., and K.H.; writing—review and editing: K.H., H.L., T.S. and B.E.W.; Visualization: P.C.; Supervision:
B.E.W.; Project Administration: P.C. and B.E.W.; Funding acquisition: B.E.W.
Funding: This research received no external funding.
Appl. Sci. 2019,9, 4432 13 of 14
Acknowledgments:
We would like to thank the North Carolina Department of Agriculture and Consumer Services
and Ryes Greenhouses for assistance with this research.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. US Congress. Agricultural Act of 2014; US Government Printing Office: Washington, DC, USA, 2014.
2. US Congress. Agricultural Act of 2018; US Government Printing Office: Washington, DC, USA, 2018.
3.
Il’in, S.G. The effect of mineral nutrition on the formation of essential oils in the plant. Tekhnicheskie Kul’tury
1940,1, 87–98.
4.
B
ó
csa, I.; M
á
th
é
, P.; Hangyel, L. Effect of nitrogen on tetrahydrocannabinol (THC) content in hemp
(Cannabis sativa L.) leaves at different positions. J. Int. Hemp Assoc. 1997,4, 80–81.
5.
Coffman, C.B.; Gentner, W.A. Responses of greenhouse-grown Cannabis sativa L. to nitrogen, phosphorus,
and potassium. Agron. J. 1977,69, 832–836.
6.
Coffman, C.B.; Gentner, W.A. Cannabinoid profile and elemental uptake of Cannabis sativa L. as influenced
by soil characteristics. Agron. J. 1975,67, 491–497.
7.
Taura, F.; Morimoto, S.; Shoyama, Y.; Mechoulam, R. First direct evidence for the mechanism of. DELTA.
1-tetrahydrocannabinolic acid biosynthesis. J. Am. Chem. Soc. 1995,117, 9766–9767. [CrossRef]
8.
Taura, F.; Sirikantaramas, S.; Shoyama, Y.; Yoshikai, K.; Shoyama, Y.; Morimoto, S. Cannabidiolic-acid synthase,
the chemotype-determining enzyme in the fiber-type Cannabis sativa.FEBS Lett.
2007
,581, 2929–2934. [CrossRef]
[PubMed]
9.
Bryson, G.M.; Mills, H.A.; Sasseville, D.N.; Jones, J.B., Jr.; Barker, A.V. Plant Analysis Handbook III: A Guide to
Sampling, Preparation, Analysis and Interpretation for Agronomic and Horticultural Crops; Micro-Macro Publishing,
Inc.: Athens, GA, USA, 2014.
10.
Landis, H.; Hicks, K.; Cockson, P.; Henry, J.B.; Smith, J.T.; Whipker, B.E. Expanding leaf tissue nutrient survey
ranges for greenhouse cannabidiol-hemp. Crop Forage Turfgrass Manag. 2019,5, 1–3. [CrossRef]
11.
Hoagland, D.R.; Arnon, D.I. The water-culture method for growing plants without soil. Circ. Calif. Agric.
Exp. Stn. 1950,347, 32, (2nd ed.).
12.
Heslop-Harrison, J.; Heslop-Harrison, Y. Studies on Flowering-Plant Growth and Organogenesis: III. Leaf
Shape Changes Associated with Flowering and Sex Differentiation in Cannabis sativa. In Proceedings of
the Royal Irish Academy. Section B: Biological, Geological, and Chemical Science; Royal Irish Academy: Dublin,
Ireland, 1957; Volume 59, pp. 257–283.
13.
Mediavilla, V.; Jonquera, M.; Schmid-Slembrouck, I.; Soldati, A. Decimal code for growth stages of hemp
(Cannabis sativa L.). J. Int. Hemp Assoc. 1998,5, 68–74.
14.
NCDA&CS. Plant, Waste, Solution, and Media Analytical Procedures. North Carolina Department of
Agricultural and Consumer Services Agronomic Division, 2015. Available online: www.ncagr.gov/agronomi/
documents/PWSMMethodology.pdf (accessed on 23 September 2019).
15.
Barnes, J.; Whipker, B.; McCall, I.; Frantz, J. Nutrient Disorders of ‘Evolution’ Mealy-cup Sage. HortTechnology
2012,22, 502–508. [CrossRef]
16.
Barnes, J. Characterization of Nutrient Disorders of Floriculture Species. Master ’s Thesis, North Carolina
State University, Raleigh, NC, USA, 1 May 2017.
17. Barker, A.V.; Pilbeam, D.J. Handbook of Plant Nutrition; CRC Press: Boca Raton, FL, USA, 2007.
18.
Gibson, J.L.; Pitchay, D.S.; Williams-Rhodes, A.L.; Whipker, B.E.; Nelson, P.V.; Dole, J.M. Nutrient Deficiencies
in Bedding Plants; Ball Publishing: Batavia, NY, USA, 2007.
19.
Van der Werf, H.M.G. Agronomy and Crop Physiology of Fiber Hemp: A Literature Review; No. 142; CABO:
Waageningen, The Netherlands, 1991.
20.
Yruela, I. Copper in plants: Acquisition, transport and interactions. Funct. Plant Biol.
2009
,36, 409–430.
[CrossRef]
21.
Hu, H.; Brown, P.H. Localization of boron in cell walls of squash and tobacco and its association with
pectin (evidence for a structural role of boron in the cell wall). Plant Physiol.
1994
,105, 681–689. [CrossRef]
[PubMed]
Appl. Sci. 2019,9, 4432 14 of 14
22.
Whittington, W.J. The role of boron in plant growth: II. the effect on growth of the radicle. J. Exp. Bot.
1959
,
10, 93–103. [CrossRef]
23. Haydon, C.F. Boron toxicity of strawberries. Commun. Soil Sci. Plant Anal. 1981,12, 1085–1091. [CrossRef]
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
