PINE BARK RATIO IN SUBSTRATE FOR CITRUS ROOTSTOCK NURSERY PRODUCTION

Abstract

Fruit tree cultivation requires rootstocks that are resistant to both biotic and abiotic stresses. The container size and substrate used are essential components in their development. Despite this, there are few studies on the impact of substrates on plant development in citrus trees under nursery conditions. This study aimed to assess the effects of three different ratios of pine bark in the substrate of three developing citrus rootstocks in a protected environment (greenhouse). The study conducted at the Cazones Nursery in Cazones de Herrera, Veracruz, Mexico, hypothesized that an increase in bark proportion would lead to a rise in the physical and chemical characteristics of the substrate and the development of the three rootstocks. The study utilized a completely randomized design with a factorial arrangement (A × B). Factor A (rootstock) had three levels: Citrus aurantium L. (Sour Orange), C. volkameriana Pasq. (Volkamer Lemon), and C. sinensis L. × Poncirus trifoliata L. (Citrage C-35). Factor B (substrate) had four levels (0, 10, 20, and 30 % pine bark), resulting in 12 treatments with 20 repetitions each. The physical and chemical characteristics of the substrates were determined, and the plant height and stem diameter were measured. Pine bark positively affected the apparent and real densities, total porosity, electrical conductivity, and cation exchange capacity. The growth dynamics of the three rootstocks were greater during the second and third months after grafting. When grown in substrates with a total porosity of 46–54 %, Volkamer Lemon, Citrage C-35, and Sour Orange rootstocks reached a plant height of 124.1, 110.5, and 84.5 cm, respectively; the stem diameter reached 6.9 mm. Porosity and cation exchange capacity increased when pine bark was added to the substrates. By evaluating the substrates and managing them proportionally, it is possible to obtain plants suitable for grafting (with 5 to 6 mm of stem) within four months after transplanting. This results in less time spent in the nursery and reduced costs.

Full text

1
Agrociencia
1
Colegio de Postgraduados Campus Montecillo. Carretera México-Texcoco km 36.5, Montecillo,
Texcoco, State of Mexico, Mexico. C. P. 56264.
* Author for correspondence: avillega@colpos.mx
ABSTRACT
Fruit tree cultivation requires rootstocks that are resistant to both biotic and abiotic stresses.
The container size and substrate used are essential components in their development. Despite
this, there are few studies on the impact of substrates on plant development in citrus trees
under nursery conditions. This study aimed to assess the eects of three dierent ratios of
pine bark in the substrate of three developing citrus rootstocks in a protected environment
(greenhouse). The study conducted at the Cazones Nursery in Cazones de Herrera, Veracruz,
Mexico, hypothesized that an increase in bark proportion would lead to a rise in the physical
and chemical characteristics of the substrate and the development of the three rootstocks. The
study utilized a completely randomized design with a factorial arrangement (A × B). Factor A
(rootstock) had three levels: Citrus aurantium L. (Sour Orange), C. volkameriana Pasq. (Volkamer
Lemon), and C. sinensis L. × Poncirus trifoliata L. (Citrage C-35). Factor B (substrate) had four
levels (0, 10, 20, and 30 % pine bark), resulting in 12 treatments with 20 repetitions each. The
physical and chemical characteristics of the substrates were determined, and the plant height
and stem diameter were measured. Pine bark positively aected the apparent and real densities,
total porosity, electrical conductivity, and cation exchange capacity. The growth dynamics of
the three rootstocks were greater during the second and third months after grafting. When
grown in substrates with a total porosity of 46–54 %, Volkamer Lemon, Citrage C-35, and Sour
Orange rootstocks reached a plant height of 124.1, 110.5, and 84.5 cm, respectively; the stem
diameter reached 6.9 mm. Porosity and cation exchange capacity increased when pine bark was
added to the substrates. By evaluating the substrates and managing them proportionally, it is
possible to obtain plants suitable for grafting (with 5 to 6 mm of stem) within four months after
transplanting. This results in less time spent in the nursery and reduced costs.
Keywords: Citrus, growth dynamics, substrate characterization.
INTRODUCTION
In Mexico, fruit tree production in nurseries has traditionally been carried out in open
environments (Soto-Plancarte et al., 2015; Barra et al., 2016) without knowledge of the
genetic origin of the rootstock and cultivar. This propagation method increases the
Citation: Pacheco-Chacón
AG, Villegas-Monter A, Trejo-
Téllez LI, Zavaleta-Mancera
HA, Calderón-Zavala G. 2024.
Pine bark ratio in substrate
for citrus rootstock nursery
production.
Agrociencia. hps://doi.
org/ 10.47163/agrociencia.
v58i3.2959
Editor in Chief:
Dr. Fernando C. Gómez Merino
Received: February 02, 2023.
Approved: January 23, 2024.
Published in Agrociencia:
April 19, 2024.
This work is licensed
under a Creative Commons
Aribution-Non- Commercial
4.0 International license.
PINE BARK RATIO IN SUBSTRATE FOR CITRUS
ROOTSTOCK NURSERY PRODUCTION
Andrea Guadalupe Pacheco-Chacón1, Ángel Villegas-Monter1*, Libia Iris Trejo-Téllez1,
Hilda Araceli Zavaleta-Mancera1, Guillermo Calderón-Zavala1
1

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 2
risk of pest infestations and the spread of diseases. Production has been carried out in
protected spaces (greenhouse) using substrates in containers to ensure phytosanitary
quality and improve establishment and development in the eld (Haase et al., 2021).
The substrate can be of mineral origin (perlite, vermiculite, sand, tezontle, and
tepeil), organic origin (peat moss, compost, coconut ber, rice, and coee husks), or
subproducts of industrial-forestry activities such as pine bark. To achieve the physical
and chemical characteristics required for optimal plant development in containers,
nurseries use three to four material mixtures. These are designed to provide the
necessary porosity, electrical conductivity, moisture retention, particle size, pH, and
cation exchange capacity (Ceccagno et al., 2019).
In Mexico, due to its physical and chemical characteristics, peat is commonly used
as the main component in forestry and ornamental nurseries, along with agrolite
and vermiculite (Fascella, 2015). However, pine bark could replace peat, which is
imported and extracted from natural sources (Urák et al., 2017). Pine bark increases
total porosity, water availability, and nutrient absorption when mixed with organic
and mineral components. This favors root development and plant growth and reduces
damage from pathogens by not providing ideal conditions for their development and
proliferation (Altland et al., 2014).
Rootstocks are utilized in fruit trees to hasten and enhance production, provide pest
and disease tolerance, and improve fruit quality (Shafqat et al., 2019). The agronomic
and phytosanitary quality of the plant is critical for its subsequent behavior in the eld.
Similarly, the substrate and bag size components are crucial to plant development
in the nursery (Correa-Moreno et al., 2022). This study aimed to assess the impact
of dierent proportions of pine bark in the substrate on the growth of three citrus
rootstocks during the nursery stage under greenhouse conditions. The hypothesis is
that increasing the proportion of pine bark will improve the physical and chemical
characteristics of the substrate, leading to enhanced development of the rootstocks.
MATERIALS AND METHODS
Study area location
The research occurred between June 6 and December 6, 2021, in a protected area
within the certied Cazones Plant-Producing Nursery (UPC 30033065/2016) in
Cazones de Herrera, Veracruz, Mexico (20° 40’ N, 97° 28’ W, at an altitude of 10 m).
The climate is tropical, warm, and subhumid, with an average annual rainfall of 1200
mm and an average yearly temperature of 25 °C. The experiment utilized plants from
a certied seed-producing orchard (3033066/2016) at the Cazones nursery. The plants
were germinated in plastic trays measuring 45 x 32 x 12 cm with a peat, perlite, and
agrolite substrate in a 6:2:2 ratio (v:v:v). Each seed was sown individually, with the
hilum facing down. When the plants had between two and four true leaves, they were
transplanted into 120 mL tubes with the same substrate. Additionally, Osmocote Plus®
fertilizer (15N-9P-12K) was added, which has a release period of 5 to 6 months.

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 3
Plant material and treatments
The study utilized three-month-old rootstock plants derived from seeds: 80 from
Sour Orange (Citrus aurantium L.), 80 from Volkamer Lemon (C. volkameriana Pask.),
and 80 from Citrange C-35 (C. sinensis L. × Poncirus trifoliata L.). The plants measured
21.26 cm and 1.78 mm, 27.14 cm and 2.03 mm, and 30.67 cm and 2.05 mm in height
and stem diameter, respectively. Subsequently, they were transplanted into 4 L black
polyethylene bags.
Four substrates were used in the experiment (Table 1). The rst substrate was used as
a control, which consisted of river valley soil (RV) and tepeil (Tz) obtained from the
region and used by the Cazones nursery. River valley soil is typically found in ood
plains and has a lower silt content and a higher sand percentage. This soil contains
goethite as the primary mineral (Arce and Rivera, 2018). Tepeil has a discontinuous
crystalline structure and high porosity due to the presence of clays (Vizcarra-de los
Reyes, 2020). To increase the total porosity of the control, pine bark (PB) was added to
substrates two, three, and four at 10, 20, and 30 %, respectively (Altland et al., 2014).
Table 1. Substrates used for the growth of three citrus rootstocks under
nursery conditions.
Substrate Components Proportion (v:v)
1 C (RV:Tz) 3:1
2 C:PB 9:1
3 C:PB 8:2
4 C:PB 7:3
C: control; RV: river valley soil; Tz: tepeil; PB: pine bark.
Management
During the transplanting process, the roots of the plants were trimmed to standardize
their size and facilitate their placement into the bags using number 2 pruners (FELCO®,
Swierland). To prevent the spread of diseases, both the tool and the roots were
disinfected using quaternary ammonium salts (1 g L-1) (Timsen Biologics). Osmocote
Plus® fertilizer (15N-9P-12K) with a 5–6 month release (2 g per plant) was applied.
The pine bark used in this study was sourced from Pinus patula Schiede ex Schltdl. et
Cham. and was composted for six months.
Four agrochemical applications were carried out during the research, as established
by the Cazones Nursery. These included the application of 1) Raízal 400® (a root
growth stimulant) at a concentration of 2 g L-1, 15 days after transplanting; 2) DAP®
[(NH4)₂HPO4, 18N-46P-00K, 2 g L-1]; 3) Nitrofoska® (12N-8P-16K+3MgO, 2 g L-1)
applied 90 days after transplanting; and 4) Humic + N® (13N-7P-7K, 2 mL L-1; Nasa
Agro Organics) applied 150 days after transplanting. To avoid competition with the
main shoot, lateral shoots were periodically removed. Irrigation was applied manually
every four days using a graded container: 100 mL per plant in the rst month, 350 mL
per plant in the second month, and 600 mL per plant from the third to the sixth month.

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 4
Study variables
Plant height (cm) was measured from the surface of the soil of the stem to the apex,
while diameter was measured at 10–15 cm above the soil surface every 28 days, starting
from the rst month after transplant until the end of the six-month experiment. Plant
height and stem diameter were measured using a measuring tape (Truper® FH-8M,
Mexico) and a caliper (Truper® Digital calibrator, Mexico), respectively.
Before the experiment, the physical characteristics of the substrate, including grain
size, apparent density (Ad), true density (Td), total porosity (TP), and electrical
conductivity (EC), were determined. Additionally, the chemical properties of the
substrate, including pH and cation exchange capacity (CEC), were evaluated at the
beginning and end of the research. These evaluations were conducted at the Soil
Physics and Plant Nutrition laboratories of the Soil Science Program at the College of
Postgraduates in Texcoco, State of Mexico, Mexico.
The grain size was determined on the four substrates using 500 g per sample and
seven sieves with the following mesh sizes (mm): 6.36, 4.76, 3.36, 2, 1, 0.5, 0.25, and the
receiver. The TP was determined by measuring the mass of water required to saturate a
soil sample of a known total volume. The Ad was determined using the probe method,
and the Td was determined using the water pycnometer method (Teixeira et al., 2017);
pH, EC, and CEC were measured using standard methods. The pH was measured
using a potentiometer with a substrate-to-distilled-water ratio of 1:2 (v:v). EC was
measured using a conductometer with the extract and saturation method, while CEC
was measured using the ammonium acetate method (Page et al., 1983).
Experimental design
A factorial arrangement A × B was utilized, where factor A represented the three
rootstocks and factor B represented the four substrates. The substrates included three
incorporating pine bark at 10, 20, and 30 %, and a control without pine bark. Each of
the 12 resulting treatments had 20 repetitions (240 experimental units), distributed
completely at random. The experimental unit consisted of one plant of each rootstock
(described earlier) in a black, 4 L polyethylene bag. Analyses of variance and mean
tests were conducted (Tukey, p ≤ 0.05) for the variables plant height (cm) and stem
diameter (mm). The data were processed using R-4.2.1® software.
RESULTS AND DISCUSSION
Physical characteristics of the substrates before the experiment
Grain size
The substrates used in the experiment exhibited varying grain sizes (Table 2), resulting
in dierent porosity levels. This, in turn, directly aects the availability of water and
oxygen for the roots of the established plants.

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 5
Table 2. Physical characteristics of the four substrates used to assess the eects of dierent ratios
of pine bark on the development of citrus rootstocks, measured before the experiment.
Substrate*
Grain size (%) Ad Td TP
Mesh number Receiver
6.36 4.76 3.36 2 1 0.5 0.25 (g cm-3) (%)
1 4.3 2.1 10.4 9.8 7.1 8.4 8.9 49.0 1.26 2.4 46
2 2.2 1.7 7.4 8.3 9.0 7.3 7.3 56.7 1.23 2.3 48
3 2.8 1.5 7.9 9.7 9.9 7.3 7.5 53.2 1.19 2.2 50
4 3.3 1.3 8.5 9.0 10.4 8.0 8.4 51.1 1.05 2.1 54
RV: river valley soil; Tz: tepeil; PB: pine bark; Ad: apparent density; Td: true density; TP: total
porosity. *Substrate 1: control, RV:Tz, 3:1 (v:v); substrate 2: C:CP, 9:1 (v:v); substrate 3: C:CP, 8:2
(v:v); substrate 4: C:CP, 7:3 (v:v); n = 4.
The addition of pine bark was projected to reduce the weight of particles in the
receiver from substrate 1 to substrate 4 by 10 to 30 %. However, this trend was only
observed from substrates 2 to 4, with substrate 1 having the lowest percentage in the
receiver. This is because all meshes included a higher percentage of tepeil particles,
indicating that the pine bark particles were smaller. Schäfer and Lerner (2022) state
that for container vegetable production, substrates should consist of particles between
3.5 and 8 mm. Smaller particles promote a greater release of ions into the solution and
increase electrical conductivity.
The following results were obtained for coarse (≥ 2 mm), medium (0.5–2 mm), and
ne particles (≤ 0.5 mm in weight) (Khamare et al., 2022). Coarse particles: substrate 1
(17.3 %), substrate 2 (14.6 %), substrate 3 (14.8 %), and substrate 4 (16.4 %); medium
particles: substrate 1 (16.9 %), substrate 2 (17.3 %), substrate 3 (17.2 %), and substrate
4 (19.4 %); and ne particles: substrate 1 (16.8 %), substrate 2 (11.3 %), substrate 3
(12.2 %), and substrate 4 (13.1 %). Given the lack of specic recommendations for citrus
and the results of this study, the percentages generated could serve as a reference for
developing the three rootstocks in nursery conditions.
Apparent (Ad) and true (Td) densities
According to Martínez and Roca (2011), the apparent density for plant production
in pots should not exceed 0.75 g cm-3. The values obtained for Ad in the substrates
evaluated in this study exceeded the limit, possibly due to the inclusion of valley soil,
known for its small particles. However, these values decreased with the addition of
pine bark, as demonstrated in substrates 2 and 4. Additionally, the total porosity values
increased (Table 2). The apparent density of a substrate depends on its particle size. An
increase in apparent density can cause salinity and compaction in the substrate (Soto-
Bravo and Betancourt-Flores, 2022).
According to Martínez and Roca (2011), the density of organic materials is around
1.45 g cm-3, while minerals have a density of 2.65 g cm-3. Values within this range are
considered optimal. For the present study, the values vary from 2.1 to 2.4 g cm-3 (Table 2).

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 6
Total porosity (TP)
There are no specic recommendations regarding the total porosity of the substrate
used in citrus plants. However, it has been suggested that for poed ornamentals
and horticulture, values should range from 70 to 80 % (Sánchez-Cardozo and Díaz-
Barrera, 2019). These values have been determined on peat, vermiculite, and perlite
substrates. Fields et al. (2021) noted that pine bark TP varies depending on particle
size. Substrates with particles ≤ 16 mm, ≤ 6.3 mm, and >12.6 mm show 78, 79, and 81 %
TP, respectively. However, the values obtained (Table 2) are lower than those reported,
possibly due to 50 % of the samples being composed of valley soil, which consists of
small particles. On the other hand, substrate 1 (control) had a higher percentage of
tepeil particles in all meshes but had the lowest porosity percentage. This indicates
that incorporating pine bark improves this parameter. Based on the observed response
and the satisfactory development of the three rootstocks used, which were adequate
for grafting four months after transplant, we can conclude that they require a 46 to
54 % TP to develop in a greenhouse environment.
Electrical conductivity (EC)
Furlani et al. (2009) recommend EC values of 2–2.5 dS m-1 for citrus rootstock growth.
Bataglia et al. (2005) compared two fertilization management systems (fertigation
and slow-release fertilization) in citrus rootstocks with a pine bark and vermiculite
substrate. They found that values increased from 2 to 5 dS m-1 throughout the growth
period due to salt accumulation from fertilization. The study shows a decrease in
EC from the start to the end of the experiment for all substrates (Table 3), indicating
that adding pine bark reduced EC. However, there were no signicant eects on the
development of the three rootstocks. To avoid toxicity problems, substrates should
have a low EC and controlled nutrient concentration (Bataglia et al., 2005).
Chemical characteristics of the substrates
pH
Arce and Rivera (2018) state that the optimal pH range for producing citrus rootstocks
in pots ranges from 5.5 to 6, which maximizes nutrient availability. A slight increase
in pH values for all substrates (Table 3) was registered at the end of the experiment,
which can be aributed to irrigation, fertilization, and management. Regarding the
incorporation of pine bark, no dened tendency was observed. Therefore, we can
claim that it did not aect the pH of the substrates during the evaluation period.
However, the pH values are above the range indicated as optimum for nutritional
availability. This may be related to 50 % of the sample being composed of valley
land, which characteristically has a neutral pH (Arce and Rivera, 2018). During plant
development, substrate variations may occur due to management practices such as
irrigation, fertilization, and dierences between species (Schäfer et al., 2008). It can be
concluded that these pH values (Table 3) do not limit the development of the three
rootstocks under nursery conditions in greenhouse environments.

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 7
Cation exchange capacity (CEC)
The substrates generally had optimal CEC values, as Schäfer et al. (2008) reported,
with values greater than 20 cmolc kg-1. However, some exceptions existed, such as
substrate 2 in Sour Orange, substrate 1 in Volkamer Lemon, and substrates 2 and 3
in C-35 (Table 3). The nal CEC values were higher than the initial values, indicating
that management practices positively impacted this variable. Altland et al. (2014)
noted that the variation in results between substrates can be aributed to dierences
in the distribution of pine bark particle sizes. They evaluated the CEC in dierent
particle sizes of pine bark and found that as the percentage of coarse particles (> 2
mm) decreases, CEC values also decrease, obtaining CEC values ranging from 21.3
to 46.5 cmolc kg-1 in pine bark. This coincides with the ndings of this investigation,
where substrates 2, 3, and 4 had the lowest percentages of particle size > 2 mm and the
greatest CEC.
Plant height (cm) and stem diameter (mm)
Statistical dierences (p ≤ 0.05) were observed for the plant height and stem diameter
variables for the rootstock factor (Table 4). This nding is consistent with Girardi
et al. (2007), who reported that Volkamer Lemon exhibits greater growth under
Table 3. Chemical characteristics and electrical conductivity of four substrates used
to assess the eects of dierent ratios of pine bark on the development of citrus
rootstocks, at the beginning and the end of the experiment.
Rootstock Substrate* pH EC (dS m-1) CEC (cmolc kg-1)
Before the experiment
17.69 3.06 16.5
27.70 3.13 21.7
37.70 3.86 20.7
47.70 2.34 22.7
After the experiment
Sour
Orange
17.92 2.55 20.71
27.92 2.35 19.61
37.95 2.18 22.30
4 8.00 1.71 23.20
Volkamer
Lemon
1 8.04 2.41 18.58
2 8.05 2.35 20.12
3 8.05 1.96 22.30
47.93 1.98 23.73
Citrange
C-35
17.89 3.54 20.18
27.98 2.26 19.09
37.96 2.29 18.05
47.95 2.03 25.28
RV: river valley soil; Tz: tepeil; PB: pine bark; EC: electrical conductivity; CEC:
cation exchange capacity. *Substrate 1: control, RV:Tz, 3:1 (v:v); Substrate 2: C:CP, 9:1
(v:v); Substrate 3: C:CP, 8:2 (v:v); Substrate 4: C:CP, 7:3 (v:v); n = 4.

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 8
nursery conditions. Volkamer Lemon showed greater plant height than C-35, which
was superior to Sour Orange. Regarding stem diameter, C-35 surpassed Volkamer
Lemon, superior to Sour orange (Table 4). According to Albrecht et al. (2020), C-35
and Volkamer Lemon reach the recommended stem diameter for grafting (5 to 6 mm)
faster. This may be due to the higher number of stomata in both varieties (data not
shown), which promotes photosynthesis (Xiong and Flexas, 2020).
Between months two and three, the three rootstocks displayed a signicant increase
in height (Figure 1). There was no signicant eect on the four substrates during the
rootstock development process. However, in substrate 4, Volkamer Lemon (124.1 cm
for plant height and 7.9 mm for stem diameter) and Citrange C-35 (110.5 cm for plant
height and 8.4 mm for stem diameter) exhibited the most growth. In substrate 1, Sour
Orange showed the least growth, with a plant height of 84.5 cm and a stem diameter
of 6.9 mm (Figures 1 and 2). Meanwhile, Volkamer Lemon displayed the most growth
and consistent development throughout the evaluation period.
The growth dynamics of the tested rootstocks were similar to those of the ‘Cravo’
rootstock when grown in a pine bark-based substrate (de Almeida et al., 2012).
However, they were not similar to the Swingle Citrumelo, which exhibited the most
growth during months four and ve after transplanting (Sauer-Liberato et al., 2021).
The three rootstocks grown in all substrates reached the recommended diameter for
grafting (5 to 6 mm) four months after transplanting, as previously indicated (Albrecht
et al., 2020). Transplanting is one of the most challenging stages in a nursery, as the
roots are not fully functional in the rst few weeks; the substrate plays a crucial role
in preventing water stress. Among the citrus varieties evaluated, Volkamer Lemon
and C-35 exhibited the most growth, reaching the suggested diameter for grafting in a
shorter time than Sour Orange. This could reduce production costs.
Arrieta-Ramos et al. (2014) conducted a study on the eect of root malformation
on the growth of Citrange Carrizo, Swingle Citrumelo (C.P.B. 4475), and Volkamer
Lemon in an open environment. The substrate used was a mixture of river valley soil,
vermicompost, and agrolite in a 3:1:1 ratio (v:v:v). At the time of transplanting, the
plants were four months old. Height and stem diameter were evaluated when the
plants were 10 months old. Volkamer Lemon reached a height of 86 cm and a stem
Table 4. Plant height and stem diameter of three citrus rootstocks grown in
four substrates with dierent ratios of pine bark under nursery conditions.
Rootstock Plant height (cm)Stem diameter (mm)
Volkamer Lemon 118.05 a 7.8 b
Citrange C-35 108.56 b 8.2 a
Sour Orange 79.69 c 6.8 c
HMSD 4.28 0.23
*Means with dierent leers in each column indicate signicant statistical
dierences (Tukey, p ≤ 0.05). HMSD: honest minimum signicant dierence.
n = 20.

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 9
Figure 1. Growth changes of citrus plants grown in four substrates with dierent ratios of pine
bark under nursery conditions. A: Sour Orange; B: Volkamer Lemon; C: Citrange C-35. Substrate
1: control, RV:Tz, 3:1 (v:v); Substrate 2: C:CP, 9:1 (v:v); Substrate 3: C:CP, 8:2 (v:v); Substrate 4:
C:CP, 7:3 (v:v). RV: river valley soil; Tz: tepeil; PB: pine bark. n = 20.
0
10
20
30
40
50
60
70
80
90
Jun Jul Aug Sep Oct Nov Dec
Plant height (cm)
Substrate 1 Substrate 2 Substrate 3 Substrate 4
0
20
40
60
80
100
120
140
Jun Jul Aug Sep Oct Nov Dec
Plant height (cm)
Substrate 1 Substrate 2 Substrate 3 Substrate 4
0
20
40
60
80
100
120
Jun Jul Aug Sep Oct Nov Dec
Plant height (cm)
Substrate 1 Substrate 2 Substrate 3 Substrate 4
Y = -1.64x2 + 24.05x - 3.65, R² = 0.98
Y = -1.60x2 + 22.68x - 1.33, R² = 0.99
Y = -1.76x2 + 23.04x - 2.39, R² = 0.92
Y = -1.43x
2
+ 21.81x - 1.69, R² = 0.98
Y = -0.72x2 + 21.06x - 4.66, R² = 0.99
Y = -1.04x2 + 23.57x - 2.28, R² = 0.99
Y = -1.70x2 + 29.15x - 3.00, R² = 0.99
Y = -1.37x
2
+ 27.46x - 1.07, R² = 0.99
Y = -3.55x2 + 41.39x - 10.31, R² = 0.96
Y = -3.27x2 + 39.67x - 9.18, R² = 0.97
Y = -3.02x2 + 37.15x - 6.31, R² = 0.97
Y = -3.40x2 + 40.58x - 8.95, R² = 0.97
A
B
C

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 10
Figure 2. Stem diameter changes of citrus plants grown in four substrates with dierent ratios
of pine bark under nursery conditions A: Sour Orange; B: Volkamer Lemon; C: Citrange C-35.
Substrate 1: control, RV:Tz, 3:1 (v:v); Substrate 2: C:CP, 9:1 (v:v); Substrate 3: C:CP, 8:2 (v:v);
Substrate 4: C:CP, 7:3 (v:v). RV: river valley soil; Tz: tepeil; PB: pine bark. n = 20.
A
B
C
0
1
2
3
4
5
6
7
8
Jun Jul Aug Sep Oct Nov Dec
Stem diameter (mm)
Substrate 1 Substrate 2 Substrate 3 Substrate 4
0
1
2
3
4
5
6
7
8
9
Jun Jul Aug Sep Oct Nov Dec
Stem diameter (mm)
Substrate 1 Substrate 2 Substrate 3 Substrate 4
0
1
2
3
4
5
6
7
8
9
Jun Jul Aug Sep Oct Nov Dec
Stem diameter (mm)
Substrate 1 Substrate 2 Substrate 3 Substrate 4
Y = -0.11x2 + 1.82x – 0.19, R² = 0.98
Y = -0.10x2 + 1.65x – 0.15, R² = 0.99
Y = -0.10x2 + 1.71x – 0.18, R² = 0.96
Y = -0.09x2 + 1.60x – 0.01, R² = 0.97
Y = -0.12x2 + 1.91x – 0.21, R² = 0.98
Y = -0.12x2 + 1.96x – 0.15, R² = 0.97
Y = -0.12x2 + 1.93x – 0.34, R² = 0.99
Y = -0.12x2 + 1.93x – 0.36, R² = 0.99
Y = -3.55x2 + 41.39x – 10.31, R² = 0.96
Y = -3.27x2 + 39.67x – 9.18, R² = 0.97
Y = -3.02x2 + 37.15x – 6.31, R² = 0.97
Y = -3.40x2 + 40.58x –8.95, R² = 0.97

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 11
diameter of 7.5 mm. In our study, six months after transplanting, Volkamer Lemon
reached a height of 124 cm and a diameter of 7.9 mm. This indicates that the plants
obtained were suitable for grafting four months prior. The results demonstrate the
signicance of substrate eects, management, and environmental conditions on plants
grown in greenhouse environments.
Arce and Rivera (2018) evaluated the eect of substrates and fertilizer on Citrange
Carrizo and Citrumelo Swingle rootstocks. They used ve substrates for their growth:
1) Promix and sand in a 1:1 ratio (v:v) (control); 2) Promix + sand + coconut ber; 3)
Promix + sand + coee compost; and 4) Promix + sand + rice husk. The laer three
substrates were in a 1:1:1 ratio (v:v:v). After six months, the ‘Swingle’ plants ranged
from 31.1 to 49 cm and had a stem diameter of 3 to 4.25 mm, while Carrizo reached a
plant height between 40.3 and 66.7 cm and stem diameters between 2.66 and 4 mm.
These results are lower than those reported here. The substrate composed of rice husk
had a negative inuence on the growth of both rootstocks. ‘Carrizo’ showed the best
development in substrate 3, while ‘Swingle’ did not exhibit signicant dierences in
any substrate except for rice husk. This highlights the importance of assessing the
physical and chemical characteristics of substrates for each rootstock.
The analysis of plant height (cm) and stem diameter (mm) means in the interaction
of the studied factors (Rootstock × Substrate) showed that Volkamer Lemon had
greater growth than C-35 and Sour Orange in all substrates, specically in substrate 4.
Conversely, Citrange C-35 in substrate 4 had the greatest stem diameter (Table 5). Sour
Table 5. Plant height (cm) and stem diameter (mm) of three citrus rootstocks
for (Rootstock × Substrate) interaction, grown in four substrates with dierent
ratios of pine bark under nursery conditions.
Rootstock × Substrate* Plant height (cm) Stem diameter (mm)
VL in S1 115.8 ab 7.7 b
VL in S2 115.5 ab 7.8 b
VL in S3 116.9 ab 8.0 b
VL in S4 124.1 a 7.9 b
C-35 in S1 107.5 b 8.3 b
C-35 in S2 109.5 b 8.2 b
C-35 in S3 106.8 b 8.1 b
C-35 in S4 110.5 b 8.4 a
NA in S1 84.5 c 6.9 c
NA in S2 79.2 c 6.8 c
NA in S3 74.6 c 6.8 c
NA in S4 80.5 c 6.7 c
HMSD 12.0 0.65
LV: Volkamer Lemon; C-35: Citrange C-35; AO: Sour Orange; S: substate; RV:
river valley soil; Tz: tepeil; PB: pine bark. *S1: Control, RV:Tz, 3:1 (v:v); S2:
C:CP, 9:1 (v:v); S3: C:CP, 8:2 (v:v); S4: C:CP, 7:3 (v:v). Means with dierent
leers in the same column are statistically dierent (Tukey, p ≤ 0.05). HMSD:
honest minimum signicant dierence. n = 20.

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 12
Orange had the least height and stem diameter, regardless of the substrate. Sauer-
Liberato et al. (2021) found that Swingle Citrumelo (Citrus paradisi Macf. × Poncirus
trifoliata L.) had a plant height of 106.8 cm and a stem diameter of 8 mm after ve
and a half months of being transplanted into 4 L polyethylene bags. The substrate
was composed of varying proportions of peat, vermiculite, and rice husk. Similar
results were obtained in this investigation with Volkamer Lemon, which had a plant
height of 124 cm and a stem diameter of 7.9 mm six months after transplanting. The
observed dierences in rootstock height are aributed to the species, as evidenced by
the growth dynamics of the three rootstocks (Figures 1 and 2).
CONCLUSIONS
The growth of Volkamer Lemon, Citrange C-35, and Sour Orange trees was enhanced
in substrates with 46 to 54 % total porosity. After four months of transplanting, the
trees were suitable for grafting. Adding pine bark at 10, 20, and 30 % of the substrate
improved its apparent and real physical densities, electrical conductivity, total
porosity, and cation exchange capacity. Under nursery conditions, Volkamer Lemon,
Citrange C-35, and Sour Orange trees can grow in substrates with pH values ranging
from 7.7 to 8.05.
ACKNOWLEDGEMENTS
The authors thank the National Humanities, Science and Technology Council (Consejo Nacional
de Humanidades, Ciencia y Tecnología - CONAHCyT) for providing the funds for A.G.P.-C’s
Master’s Degree.
Additionally, the authors thank the Cazones Nursery for providing the necessary resources to
conduct this research in a greenhouse environment.
REFERENCES
Albrecht U, Bodaghi S, Meyering B. 2020. Inuence of rootstock propagation method on
traits of grafted sweet orange trees. HortScience 55 (5): 729–737. hps://doi.org/10.21273/
hortsci14928-20
Altland JE, Locke JC, Krause AR. 2014. Inuence of pine bark particle size and pH on cation
exchange capacity. HortTechnology 24 (5): 554–559. hps://doi.org/10.21273/horech.24.5.554
Arce S, Rivera D. 2018. New media components and fertilization to accelerate the growth of
citrus rootstocks grown in a greenhouse. Horticulturae 4 (2): 10. hps://doi.org/10.3390/
horticulturae4020010
Arrieta-Ramos BG, Villegas-Monter A, Rodríguez-Mendoza MN, Luna-Esquivel G. 2014.
Desarrollo en vivero de portainjertos de cítricos con malformación de raíz. Revista Chapingo
Serie Horticultura 20 (1): 29–39. hps://doi.org/10.5154/r.rchsh.2012.06.034
Barra PJ, Inostroza NG, Mora ML, Crowley DE, Jorquera MA. 2016. Bacterial consortia
inoculation mitigates the water shortage and salt stress in avocado (Persea americana Mill.)
nursery. Applied Soil Ecology 111: 39–47. hps://doi.org/ 10.1016/j.apsoil.2016.11.012

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 13
Bataglia OC, Quaggio JA, Ferreira de Abreu M, Ronchini-Boaventura PS. 2005. Nutrient uptake
and leaching on citrus nursery production in substrate with two fertilizer management
programs. Acta Horticulturae 697: 281–284. hps://doi.org/10.17660/ActaHortic.2005.697.34
Ceccagno H, Dutra-de Souza PV, Schäfer G, Demari-Avrella E, Sidnei-Fior C, Schwarz SF.
2019. Potential of Pinus sp. needles for use as substrate conditioners in the production of
‘Trifoliata’ rootstock in greenhouses. Revista Chapingo Serie Horticultura 25 (1): 5–16.
hps://doi.org/10.5154/r.rchsh.2018.02.003
Correa-Moreno DL, Martínez MF, Acosta G. 2022. Sustratos para la producción de portainjertos
en ambiente protegido. In Martínez MF, Murcia-Riaño N, Barreto-Rojas JA, Acosta-Herrera
GH, Jaramillo-Laverde A, Correa-Moreno DL, López-Galé Y, Rodríguez-Mora D, Kondo
T, Palacios-Joya L, Beltrán-López HD. (eds.), Bases Tecnológicas para la Producción de
Material de Siembra de Alta Calidad de las Especies Cítricas para Colombia. Corporación
Colombiana de Investigación Agropecuaria (AGROSAVIA): Mosquera, Colombia, pp: 87–
105. hps://doi.org/10.21930/agrosavia.manual.7405682
de Almeida LVB, Sales-Marinho C, De Almeida-Muñiz R, Cordero de Carvalho AJ. 2012.
Disponibilidade de nutrientes e crescimento de porta-enxertos de citros fertilizados com
fertilizantes convencionais e de liberação lenta. Revista Brasileira de Fruticultura, Jaboticabal
34 (1): 289–296. hps://doi.org/10.1590/S0100-29452012000100038
Fascella G. 2015. Growing substrates alternative to peat for ornamental plants. In Asaduzzaman
M. (ed.), Soilless Culture: Use of Substrates for the Production of Quality Horticultural
Crops. Intech Open: London, UK, pp: 47–51. hps://doi.org/10.5772/59596
Fields JS, Owen JJS, Altland JE. 2021. Substrate stratication: Layering unique substrates within
a container increases resource eciency without impacting growth of shrub rose. Agronomy
11 (8): 1454. hps://doi.org/10.3390/agronomy11081454
Furlani PR, Zanei M, Bataglia OC. 2009. Citrus nursery production in soilless culture. Acta
Horticulturae 843: 255–260. hps://doi.org/ 10.17660/ActaHortic.2009.843.33
Girardi EA, Mourão F de AA, Kluge RA. 2007. Eect of seed coat removal and controlled-release
fertilizer application on plant emergence and vegetative growth of two citrus rootstocks.
Fruits 62 (1): 13–19. hps://doi.org/10.1051/fruits:2006044
Haase DL, Bouzza K, Emerton L, Friday JB, Lieberg B, Aldrete A, Davis AS. 2021. The high cost
of the low-cost polybag system: A review of Nursery Seedling Production Systems. Land 10
(8): 826. hps://doi.org/10.3390/land10080826
Khamare Y, Marble SC, Altland JE, Pearson BJ, Chen J, Devkota P. 2022. Eect of substrate
stratication without ne pine bark particles on growth of common nursery weed species
and container-grown ornamental species. HortTechnology 32 (6): 491–498. hps://doi.
org/10.21273/horech05113-22
Martínez PF, Roca D. 2011. Sustratos para el cultivo sin suelo. Materiales, propiedades y manejo.
In Flórez RVJ. (ed.), Sustratos, Manejo del Clima, Automatización y Control en Sistemas
de Cultivo sin Suelo. Editorial Universidad Nacional de Colombia: Bogotá, Colombia, pp:
37–77.
Page AL, Miller RH, Keeney DR. 1983. Methods of soil analysis, part 2: Chemical and
microbiological properties. Soil Science Society of America: Madison, WI, USA. hps://doi.
org/10.2134/agronmonogr9.2.2ed
Sánchez-Cardozo J, Díaz-Barrera L. 2019. Evaluación de sustratos elaborados a partir de residuos
celulósicos para la propagación de ores ornamentales y hortalizas. Bioagro 31 (1): 45–54.

Agrociencia 2024. DOI: hps://doi.org/10.47163/agrociencia.v58i3.2959
Scientic article 14
Sauer-Liberato EM, Leonel S, Souza JMA, Napoleão GM. 2021. Substrate mixing formulations
for citrus nursery management. Nativa 9 (5): 500–507. hps://doi.org/10.31413/nativa.
v9i5.12777
Schäfer G, de Souza PVD, Koller OC, Schwarz SF. 2008. Physical and chemical properties of
substrates to cultivate seedling of citrus rootstocks. Communications in Soil Science and
Plant Analysis 39 (7–8): 1067–1079. hps://doi.org/10.1080/00103620801925547
Schäfer G, Lerner BL. 2022. Physical and chemical characteristics and analysis of plant substrate.
Ornamental Horticulture 28 (2): 181–192. hps://doi.org/10.1590/2447-536X.v28i2.2496
Shafqat W, Jaskani MJ, Maqbool R, Khan AS, Ali Z. 2019. Evaluation of citrus rootstocks against
drought, heat and their combined stress based on growth and photosynthetic pigments.
International Journal of Agriculture and Biology 22 (5): 1001–1009.
Soto-Bravo F, Betancourt-Flores A. 2022. Evaluación de metodologías para determinar las
características físicas de un sustrato de bra de coco. Agronomía Costarricense 46 (2): 29–45.
hps://doi.org/10.15517/rac.v46i2.52044
Soto-Plancarte A, Santillán-Mendoza R, Fernández-Pavía SP, Ploe RC, Freeman S, Ortega-
Arreola R, Osuna-Ávila P, Velázquez-Monreal JJ, Rodríguez-Alvarado G. 2015. Mango
nurseries as sources of Fusarium mexicanum, cause of mango malformation disease in central
western Mexico. Phytoparasitica 43 (4): 427–435. hps://doi.org/10.1007/s12600-015-0471-4
Teixeira PC, Kangussu-Donagemma G, Fontana A, Gerlades-Teixeira W. 2017. Manual de
métodos de análise de solo (Third edition). Embrapa: Brasilia, Brazil. 574 p.
Urák I, Hartel T, Gallé R, Balog A. 2017. Worldwide peatland degradations and the related
carbon dioxide emissions: the importance of policy regulations. Environmental Science and
Policy 69: 57–64. hps://doi.org/10.1016/j.envsci.2016.12.012
Vizcarra-de los Reyes M de los A. 2020. Lecciones para la recuperación de saberes tradicionales:
Las tapias de la región de Tepeyahualco, México. Journal of Traditional Building. Architecture
and Urbanism 1: 434–446.
Xiong D, Flexas J. 2020. From one side to two sides: the eects of stoma distribution on
photosynthesis. New Phytologist 228 (6): 1754–1766. hps://doi.org/10.1111/nph.16801
AgrocienciaAgrociencia
AgrocienciaAgrociencia