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Effect of Different Enriched Vermicomposts, Humic Acid Extract and Indole-

3-Acetic Acid Amendments on the Growth of Brassica napus

Article in Plants · January 2022

DOI: 10.3390/plants11020227

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 plants
Article
Effect of Different Enriched Vermicomposts, Humic Acid
Extract and Indole-3-Acetic Acid Amendments on the Growth of
Brassica napus
Arash Hemati 1, *, Hossein Ali Alikhani 2 , Ladan Ajdanian 3 , Mehdi Babaei 3 , Behnam Asgari Lajayer 1
and Eric D. van Hullebusch 4, *

1 Department of Soil Science, Faculty of Agriculture, University of Tabriz, Tabriz 5166616422, Iran;
[email protected]
2 Department of Soil Science, University College of Agriculture and Natural Resources, University of Tehran,
Tehran 1417466191, Iran; [email protected]
3 Department of Horticultural Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad,
Mashhad 9177948974, Iran; [email protected] (L.A.); [email protected] (M.B.)
4 Institut de Physique du Globe de Paris, Université de Paris, CNRS, F-75005 Paris, France
* Correspondence: [email protected] (A.H.); [email protected] (E.D.v.H.)

Abstract: Humic acid (HA) is a specific and stable component of humus materials that behaves
similarly to growth stimulants, esp. auxin hormones, contributing to improving growth indices and
performance of plants. As a rich source of HA, vermicompost (VC) is also a plant growth stimulating





 bio-fertilizer that can enhance growth indices and performance in plants. The purpose of the
Citation: Hemati, A.; Alikhani, H.A.; present study is to compare the influence of VC enriched with bacterial and/or fertilizer, commercial
Ajdanian, L.; Babaei, M.; Asgari humic acid (CHA) extract, and indole-3-acetic acid (IAA) on improving growth characteristics and
Lajayer, B.; van Hullebusch, E.D. performance of rapeseed under greenhouse conditions. The results showed the complete superiority
Effect of Different Enriched of VC over the CHA and IAA (approximately 8% increase in the dry weights of root and aerial
Vermicomposts, Humic Acid Extract organ and nearly three times increase in seed weight). The highest values of these indices were
and Indole-3-Acetic Acid
obtained with VC enriched with Nitrogen, Sulfur, and Phosphorus, Azotobacter chroococcum and
Amendments on the Growth of
Pseudomonas fluorescens; the lowest value was obtained with VC enriched with urea. Additionally, the
Brassica napus. Plants 2022, 11, 227.
application of 3% VC and the control involved the highest and lowest values in all traits, respectively.
https://doi.org/10.3390/
The SPAD (chlorophyll index) value and stem diameter were not significantly affected by different
plants11020227
application levels of VC. Overall, the applications of IAA and the CHA were not found to be suitable
Academic Editors: Federico and therefore not recommended.
A. Gutiérrez-Miceli, Luc Dendooven
and Dimitris L. Bouranis
Keywords: growth stimulant; rapeseed; plant growth performance; bacteria; vermicompost
Received: 26 November 2021
Accepted: 13 January 2022
Published: 16 January 2022
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in Oil seeds are the second largest source of nutrients after cereals. In addition to being
published maps and institutional affil- rich in fatty acids, these products contain proteins as well [1]. Rapeseed is known as one of
iations. the most important oil seed plants in the world; accordingly, it is considered as the third
largest source of vegetable oil after soybean and palm [2].
Vermicompost (VC) is a plant growth stimulating organic fertilizer being a rich source
of humic acid (HA), that can improve plant growth performance indices [3]. Vermicompost
Copyright: © 2022 by the authors. contains enzymes and natural growth stimulants that, along with nutrients and HA, pro-
Licensee MDPI, Basel, Switzerland. mote plant growth and yield [4]. The HA obtained from VC is capable of competing with
This article is an open access article its commercial counterpart that are often produced from coal or leonardite, and even indole
distributed under the terms and
phytohormones (esp. IAA) [5]. Several studies have been conducted regarding the effect
conditions of the Creative Commons
of VC application on the growth and development of crops [6–8]. For instance, for Maize
Attribution (CC BY) license (https://
(Zea mays), it has been shown that VC increased plant growth yield by creating favorable
creativecommons.org/licenses/by/
conditions for plant nutrition [9]. It has also been reported that VC usually contains more
4.0/).

Plants 2022, 11, 227. https://doi.org/10.3390/plants11020227 https://www.mdpi.com/journal/plants

Plants 2022, 11, 227 2 of 19

nutrients than plant-derived organic matter, and large amounts of these nutrients have
been converted into forms that are easily absorbed by plants [10]. On the other hand, VC
has high microbial and enzymatic activity and contains large amounts of plant growth
regulators [11]. Continuous and adequate use of VC along with proper management can
increase organic carbon storage and retain water in soils. Moreover, by improving VC phys-
ical properties, it can have a beneficial effect on the growth and yield of plant species [9]. It
has also been observed that by increasing the amount of VC in the culture medium, the
amount of elements such as zinc, calcium, and nitrogen significantly increase in the plant
aerial parts [12]. Furthermore, with the application of VC in field conditions, an increase
in barley (Hordeum vulgare) yield was observed [13]. Vermicompost treatment in chickpea
(Cicer arietinum) and pea (Pisum sativum) significantly increased morphological traits such
as root length, stem length, and the number of leaves [14–16]. It was also reported that the
application of VC enriched with other nutrients compared to their separate application
was accompanied by improved morphological and physiological traits of the corn plant
(Zea mays) [17].
One of the practical drawbacks of VC is its high-volume consumption (at least 2 to
5 tons per hectare). To solve this problem, in some studies, the population change of
microorganisms in VC and the improvement of the quality of organic fertilizers have been
tested recently [18]. In addition to nitrogen fixation, some nitrogen-fixing bacteria also
dissolve insoluble mineral phosphates by producing organic acids [19,20]. A number of
diazotrophic bacteria such as Pseudomonas, Burkholderia, Agrobacterium, Azotobacter and
Ervinia are able to increase absorbable phosphorus and bio-stabilize nitrogen. Increasing the
bioavailability of phosphorus by these microorganisms is achieved by producing organic
acids that increase the amount of absorbed mineral phosphorus [21]. Thiobacillus-inoculated
VC has positive effects on the conversion of phosphate soil to absorbable phosphorus, and
Burkholderia and Herbaspirillum genus strains are also reported to increase absorbable
phosphate [22,23]. Inoculation of nitrogen-fixing microorganisms increases the amount of
nitrogen in VC and inoculation of phosphate-solubilizing microorganisms in VC with the
presence of phosphate soil and even without phosphate soil increased phosphorus in VC.
However, the direct application of phosphate soil was not particularly useful in natural
soils [24–26]. Enrichment of compost with ammonium sulfate and urea by adding nitrogen
in solid or soluble forms at the beginning of composting process has increased the total
nitrogen and increased efficiency in the field of plant growth [27,28].
Humic acid is one of another best plant growth stimulant. This substance is formed by
the decomposition of organic matters, especially the plant-derived ones, and is found in
soil, coal and peat. Humic acid that is a mixture of very large molecules with the ability
to complex metallic elements, is one of the most important components of humus [29].
Consuming HA improves cation exchange capacity and aeration by creating a layer on
soil particles and it increases root growth [30]. The effect of HA on spring wheat (Triticum
aestivum) yield has been investigated by Dinçsoy et al. [31]. The results showed that HA
increased the access to phosphorus and other nutrients and also caused a significant yield
increment. Experiments carried out on different plants have displayed that HA increases
plant growth directly and indirectly, and in different amounts for various plants [30,32]. The
direct and positive effect of HA on the growth of wheat [33], chickpeas (Cicer arietinum) [34]
and chicory (Cichorium intybus) [35] have been reported. The plant growth response curve
related to HA treatment showed that with increasing HA concentration, plant growth
increased [36]. This stimulatory effect at low concentrations could be more related to the
direct effect on the plants, which was the effect of natural hormones, along with the indirect
effect on the metabolism of soil microorganisms, the dynamics of nutrient uptake from the
soil, and the physical condition of the soil [8]. The most important and abundant auxin in
plants is IAA, which in general, depending on its concentration, can have different effects
on plant growth [37]. The evaluation of the effect of IAA (2000, 1000 and 3000 mg L−1 )
on the improvement of Balanites aegyptiaca growth for 24 h, showed a significant increase
Plants 2022, 11, 227 3 of 19

in the height, number of branches and leaves, chlorophyll content and dry weight of the
plant [38].
Considering the importance of rapeseed in oil production and the international soci-
eties’ approach to the conservation of natural resources in line with reducing the use of
chemical fertilizers, the present study was designed and implemented on the application of
bio-fertilizers including VC as a rich source of HA. To this end, different vermicomposts
(VCs) enriched with nitrogen, NSP (Nitrogen, Sulfur, Phosphorus) chemical elements, and
Pseudomonas fluorescens and Azotobacter chroococcum at various levels were compared with
the commercial humic acid (CHA) and the IAA hormone (as substances that are naturally
present in VC). The use of enriched VC aims at reducing the amount of organic materials
to be spread on agricultural lands. On the other hand, the quasi-hormonal properties of
some compounds contained in VC and its HA content which are expected to stimulate
plant growth requires the comparison of different amendment combinations (hormonal
substances and HA to be compared with VC amendments enriched with different chemical
and biological agents) needs to be investigated. Such an approach has not been tested
before and, therefore, represents the novel aspect in this research.

2. Materials and Methods

2.1. VC Production and Selecting Enrichment Treatments
Vermicompost was produced from cow manure raw materials and plant residues in a
1:3 ratio (weight: weight) in the presence of composting worms (Eisenia fetida) for a period
of 5 months in the VC Education and Research Station of Agriculture and Natural Resources
Campus in Tehran University. To this aim, first cow manure and plant residues were placed
under sunshine for 1 month; then, small dome-shaped hills with a width of 70, length of
200, and height of 50 cm were formed. Following sufficient irrigation monitored by the
occurrence of leachate, Eisenia fetida worms were inserted into the bed (500 earthworms
per 100 kg of bed). The humidity of the hills was approximately 50–60% during the
composting period, and the humidity was maintained through daily irrigation. By the
end of the processing period, earthworms were separated from the final product (VC).
The characteristics of the VC were analyzed and are presented in Table 1 [39]. In order to
enrich VC with bacterial treatments, phosphate solubilizing bacteria (Pseudomonas) and
nitrogen-fixating bacteria (Azotobacter) were used. Azotobacter and Pseudomonas belong to
the Azotobacter chroococcum and Pseudomonas fluorescens species, respectively. The entire
used strains were obtained from the beneficial terricolous microorganism gene bank of the
department of soil science engineering at Tehran University which have been identified,
separated, and maintained in studies conducted in previous years [40]. After the second
bacterial enrichment, the inoculated bacterial population was adjusted at 4 × 109 cfu
(Colony Forming Unit) mL−1 and 25 mL of each liquid enrichment was used for inoculation
per 1 kg of VC [23]. In addition, it was attempted for this study to make use of necessary
chemical elements with the highest degrees of importance and consumption in agriculture
(nitrogen, phosphorus, and sulfur) as fertilizer treatments to enrich VC. One percent of each
element was added to VC; the source of added nitrogen was urea, the source of phosphorus
was triple superphosphate and the source of sulfur was elemental sulfur. Potassium was
not added due to the richness of potassium in the produced VC. Prior to enrichment, VC
samples were screened using a 2 mm sieve and their large particles were separated.

Table 1. Initial vermicompost properties.

EC Total N OC P K Na Fe Ca
pH C/N
(dS·m−1 ) (%) (%) (%) (%) (%) (%) (%)
7.63 2.14 1.2 24.37 0.82 6.52 1.1 0.57 8.5 20.3
Plants 2022, 11, 227 4 of 19

2.2. Soil Amendment Composition and Application Level

Factors investigated in this experiment included 4 VC fertilizer treatments at 4 fertiliz-
ing levels including indole-3-acetic acid (IAA) was provided by the University of Tehran
(Forbes Pharmaceuticals company, Maharashtra, India); 75% pure CHA from Leonardite
imported from China (Hebei China Company, Hebei, China) was obtained from the market.
Vermicompost was completely mixed with the soil at 4 levels of 0, 1, 2, and 3% (equivalents
to 0, 30, 60 and 90 g pot−1 ). Additionally, the CHA at 4 levels of 0, 200, 400, and 600 mg/kg
and IAA acid with concentrations of 10−6 , 10−5 , and 10−4 molar, 100 mL per pot (Table 2)
were used in three 10, 20, and 30 day stages as well as two flowering and reproductive
stages through hilling. The concentrations used to study was selected based on previous
data published in the literature for CHA [5,41–43] and IAA [44,45]. The use of HA and IAA
were both accumulated around the crowns of plants and the added VC was completely
mixed with pot soil.

Table 2. Soil amendment composition and application level (1- VC without enrichment (VC); 2- VC
enriched with 1% nitrogen (VC-N); 3- VC enriched with 1% nitrogen, 1% sulfur, and 1% phosphorus
(VC-NSP); 4- VC enriched with Azotobacter chroococcum (21Az) + Pseudomonas fluorescens (Ps 59)
(VC-AS); 5- Commercial humic acid (CHA); 6- Indole-3-acetic acid (IAA)).

Level 1 Level 2 Level 3 Level 4

No additive of VC 1% VC 2% VC 3% VC
No additive of VC-N 1% VC-N 2% VC-N 3% VC-N
No additive of VC-NSP 1% VC-NSP 2% VC-NSP 3% VC-NSP
No additive of VC-AS 1% VC-AS 2% VC-AS 3% VC-AS
No additive of IAA 10−6 molar of IAA 10−5 molar of IAA 10−4 molar of IAA
No additive of CHA 200 mg kg−1 CHA 400 mg kg−1 CHA 600 mg kg−1 CHA

2.3. Plant Growth and Material

Pots were placed in the greenhouse environment after being prepared. The growing
stage was carried out by manually placing five shrubs in plastic pots containing 3 kg of
soil from 20 April to 22 September 2012. The loamy soil used in this study was prepared
from the research farm of Karaj Soil and Water Institute located in Meshkin Dasht, Karaj.
Sampling was performed from a depth of 0–30 cm of the soil surface and the samples
were transferred to the laboratory. Some physicochemical properties of the soil sample
were measured after air drying, crushing, and passing through a 2 mm sieve, and are
presented in Table 3. Overall, the measurements of available P was done by the Olsen
method, total nitrogen was done by the Kjeldahl method, available K by using 1 N acetate
ammonium, available Zn and Fe estimated by DTPA-TEA extraction method, EC and pH
in saturated soil extract, and soil texture by the hydrometric method [46,47]. Generally, this
experiment was conducted based on analysis of soil used in this study. Malakouti et al. [48]
reported that the critical levels of N, P and K varies between 1000 and 2000, 25 and 450 and
8 and 15 mg kg−1 soil, respectively, based on soil characteristics. Since the soil used in the
present study showed a deficiency in N and P, the amendments used in the experiments
were enriched in N, P and/or bacterial strains; phosphate solubilizing and nitrogen-fixing
bacteria (Pseudomonas fluorescens and Azotobacter chroococcum, respectively).

Table 3. Physical and chemical properties of experimental soil.

EC Zn Fe P K N Clay
pH Sand (%) Silt (%)
(dS·m−1) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (%)
1.6 7.2 0.74 2.5 8.1 370 800 33 39 28

After drying, the soil was passed through a 4 mm sieve for use in pots. After ger-
mination and complete settlement of plant germs, their numbers were narrowed down
Plants 2022, 11, 227 5 of 19

to two per pot. In this study, the modified RGS (spring cultivar and sensitive to cold)
rapeseed cultivar was used which was obtained from Karaj Seed and Seedling Research
Institute. The minimum and maximum temperatures of the greenhouse was 20 and 28 ◦ C,
respectively, with a relative humidity of 75–80%. In addition, the rapeseed shrubs were
exposed to 14 h of light (a combination of fluorescent and tungsten lamps), daily.
The plants growth period was completed within four months during which the pots
were visited daily, and the humidity of each pot were adjusted at 0.75–0.8 field capacity in
terms of weight. The harvesting stage began by the end of the growth period, after the plants
grew clusters. After being separated from the soil and weighing the wet weight of aerial
organs, these organs were washed entirely using distilled water; next, they were placed
inside clean paper envelopes and then dried in an oven for 48 h at 65 ◦ C; subsequently,
the dry weight of the aerial organ was measured as well. Then, the dried aerial organ was
separately powdered using a grinder and then placed inside lidded containers to produce
herbal extracts and perform analytical experiments. Furthermore, the root system of the
plant was completely taken out of the soil as much as possible and then placed in a basin
full of water; next, the surrounding soil was washed away and ultimately, the wet and dry
weights of the root was measured.

2.4. Measuring Plant Characteristics after Harvesting

The height of the shrub was measured from crown to the tip of the stamen in cen-
timeters, without taking the root into account. The root was carefully separated from the
soil and washed with water to remove its surrounding soil as much as possible. After
separating the root from the soil, its length was measured in centimeters using a ruler.
All leaves for each shrub was counted during the growing and harvesting periods. The
length of the largest inflorescence in each shrub was measured in centimeters from the
stem growth location. The length of the largest inflorescence was solely measured due to
difference in the number of inflorescences under various treatments; moreover, measuring
the lengths of all inflorescences and indicating inflorescence average length would not
have represented the reality of the study in a few cases. Stem diameter was measured in
millimeters using calipers from the stem, under the first knuckle. To determine the extent
of chlorophyll in a leaf, SPAD-502 manual chlorophyll meter (Minolta, Japan) was used
without damaging plant textures and extracting from leaves. To this end, three leaves were
selected on average during the flowering stage and the extent of chlorophyll was estimated
from its central point. Leaf area was measured using a leaf area meter tool, model Delta
T-Devices UK (∆T Area Meter MK2). The extent of photosynthesis was measured in µM
CO2 × m−2 × s−1 . Stomatal conductance was measured via an aerial infrared gas analyzer
(IRGA) tool, model LCA4-ADC. At the end of the 4-month period and after the clusters
were formed and dried, the number of clusters, percentage of fertile clusters and the total
seed weight were measured for each plant according to Equation (1).

Fertile Clusters (%) = (Number of Fertile Clusters/Total Number of Clusters) × 100 (1)

2.4.1. Evaluating the Amount of Oil Contents in Rapeseed

The oil existing inside the seeds was extracted using Soxhlet method, via methanol-
chloroform organic solvent in a 1:2 ratios and three repetitions. The method was used on
rapeseed for the first time by Joshi et al. [49]. The dried seeds were powdered, transferred
to M3 Whatman Filter Paper, and weighed (Weight A). The sample was then packed tightly
and placed inside an oven for 6–8 h and was dried until reaching a stable weight (Weight B).
After cooling at room temperature in a desiccator, samples were transferred to Soxhlet pipes
and were extracted for 24 h via petroleum ether (b.p. below 50 ◦ C). Following extraction,
the packed samples were placed under a hood to evaporate the remaining petroleum ether
and become dried; ultimately, desiccator was cooled and then weighed (Weight C). Oil
contents (%) was calculated according to Equation (2).

Oil content (%) = (B − C)/(B − A) × 100% (2)

Plants 2022, 11, 227 6 of 19

The dried seeds were triturated and transferred to 3M Whatman filter paper and
weighed (weight A); the sample was tightly closed and then dried in an oven for 6–8 h until
a stable weight was reached (weight B). After cooling to room temperature in a desiccator,
the samples were transferred to Soxhlet tubes and extracted with petroleum ether for
24 h (b.p. below 50 ◦ C). Then, after extracting the packaged samples for evaporation, the
remaining petroleum ether was placed in the hood to dry and finally, the desiccator was
cooled and then weighed (weight C). Three repetitions were prepared for each sample
and the average value of these three treatments was employed to calculate the amount of
oil contents.

2.4.2. Ashing Plant Materials and Producing Herbal Extraction

The dry ashing method was employed to produce herbal extract. To this end, 1 g of
ground dry plant material was poured into a crucible and then placed inside a furnace;
temperature was gradually raised to 450 ◦ C so that white ash is produced. After the
samples were cooled, 20 mL of 2 normal hydrochloric acids was added to each sample and
then placed in a sand bath for 30 min. Finally, samples were filtered in 100 mL volumetric
flask and brought to volume [50].

2.4.3. Total Phosphorus Analysis

In order to measure phosphorus, the yellow method (Molybdovanadate) was em-
ployed. Accordingly, the plant sample solution was prepared following the preparation
of phosphorus yellow and standard solutions. First, 20 mL of the herbal extract produced
using the dry ashing method was poured into a 100 mL volumetric flask; then, 20 mL of
yellow indicator and 20 mL of distilled water were added. After 45 min, the solution was
brought to volume and phosphorus content was read using a spectrophotometer at 430 nm
wavelength. Prior to reading plant samples, the standard solutions were read using the
device and its chart was obtained [50].

2.4.4. Measuring Copper Concentration in Plant’s Aerial Organ

The concentration of copper in herbal extracts was measured and reported using an
atomic absorption spectroscopy (Shimadzu AA-670). For this assay, 0.1 g of the dried plant
organs of each pot were digested with 2 mL of 60% nitric acid overnight and then placed
in a water bath for 2 h at 90 ◦ C. After cooling, 1 mL of hydrogen peroxide was added to
the samples and the tubes were placed in a water bath at 90 ◦ C for half an hour. After
cooling, the samples were reduced to 10 mL with distilled water. Copper extracted from
the root and aerial parts of the plant were measured using a device during Shimadzu
AA-670 atomic absorption spectrometry and a calibration curve was drawn with respect to
the exclusive wavelength of each element; finally, samples were read [50].

2.5. Experiment Design and Statistical Analysis

Greenhouse experiments were performed as a factorial in the form of randomized
complete block design (RCBD) (with 2 factors including treatment and treatment level) in
four repetitions. The obtained results were analyzed using SAS software and the related
variance analysis tables were drawn. Additionally, the comparison of data mean values
was performed by using Duncan’s multiple range test at 5% level via MSTAT-C software.

3. Results and Discussion

3.1. Effects of Amendment Formulation on Morpho-physiological Characteristics of Rapeseed
3.1.1. Plant Height, Stem Diameter, and Number of Leaves
The effect of the applied soil amendments on the above traits was significant at
1% probability level (p ≤ 0.01). As indicated in Figure 1a, the highest number of leaves
was observed in VC-AS treatment at 4% application, which was significantly different
from the control and VC-N treatment. At similar levels, plant height was the highest in
VC-AS treatment, which was significantly different from all VC treatments used at this
Plants 2022, 11, 227 7 of 19

Plants 2022, 10, x FOR PEER REVIEW 8 of 20

level (Figure 1b). Additionally, the highest amount of stem diameter was observed in the
same treatment used at the 4% level, which was different from other treatments (Figure 1c).

25
(a) a a VC
20 a ab ab
bc b
Number of leaves

ab cd cd c bc VC-N
15 bc b d c
c c VC-AS
a a a a a a
10 VC-
NSP
CHA
5

0
1 2 3 4
Treatment levels

(b)
100 a
a a
ab b VC
80 b
Plant height (cm)

c
bc b b c c
c c c c
d cd
VC-N
60 a a a a a a VC-AS
40 VC-NSP
CHA
20
IAA
0
1 2 3 4

Treatment levels

(c)
12
a ab
Stem diameter (mm)

10 a b VC
ab a c
8 a a a b
c d VC-N
b b b d e
VC-AS
6 a a a a a a
VC-NSP
4
CHA
2 IAA
0
1 2 3 4

Treatment levels

Figure
Figure Comparison
1. 1. Comparisonofofaverage
averagesoil
soilapplication
applicationofofexperimental
experimentaltreatments
treatmentson onnumber
numberofofleaves
leaves(a),
(a), plant height (b) and stem diameter (c) at treatment levels (levels 1, 2, 3 and 4 for
plant height (b) and stem diameter (c) at treatment levels (levels 1, 2, 3 and 4 for VC, VC-N, VC-AS VC, VC-N, VC-
and VC-NSP treatments of 0, 1, 2 and 3% VC, 0, 200, 400 and 600 mg of CHA per kg of soil and
AS and VC-NSP treatments of 0, 1, 2 and 3% VC, 0, 200, 400 and 600 mg of CHA per kg of soil and 0,
0,6 10
10−
−6, 10−5 and 10−4 molar IAA). Different letters in each figure show significant difference at p ≤
, 10− 5 and 10−4 molar IAA). Different letters in each figure show significant difference at p ≤ 0.05
0.05 by Duncan multiple range test. VC, VC without enrichment; VC-N, VC enriched with 1% nitro-
by Duncan multiple range test. VC, VC without enrichment; VC-N, VC enriched with 1% nitrogen;
gen; VC-NSP, VC enriched with 1% nitrogen, 1% sulfur, and 1% phosphorus; VC-AS, VC enriched
VC-NSP, VC enriched
with Azotobacter with 1%
chroococcum nitrogen,
(21Az) 1% sulfur,
+ Pseudomonas and 1% phosphorus;
fluorescens (Ps 59); CHA, VC-AS, VC enriched
Commercial with
humic acid;
Azotobacter chroococcumacid.
IAA, Indole-3-acetic (21Az) + Pseudomonas fluorescens (Ps 59); CHA, Commercial humic acid; IAA,
Indole-3-acetic acid.
Plants 2022, 11, 227 8 of 19

The positive effects of VC enrichment with bacteria have been well identified [51,52].
For instance, while investigating the effect of inoculating VC with bacteria such as Azoto-
bacter chroococcum and Pseudomonas fluorescens, it was reported that inoculating of VC with
these bacteria significantly increased phosphorus and nitrogen in VC relative to the control,
which consequently lead to increased nutrient uptake and plant growth indices [53,54]. On
the other hand, it has been described that composting with the microbial community dis-
solves insoluble phosphates and thus increases the available phosphorus content [55]. Some
nitrogen-fixing bacteria, in addition to nitrogen fixing, dissolve phosphorus by producing
organic acids, which leads to increased plant growth [22]. Based on Figure 1, there was no
significant difference in leaf number and stem diameter between VC-AS and VC-NSP treat-
ments. In this regard, it could be concluded that both treatments could increase the amount
of nitrogen and phosphorus uptake by plants. However, compared to the control treatment,
which is non-enriched VC, a significant difference was observed. Additionally, the results
showed that the lowest number of leaves, height and stem diameter was observed in the
IAA treatment at the level of 2%. Compared to other concentrations of CHA used, the
application of 400 mg/kg CHA leads to the highest values in height and number of leaves
in rapeseed. The use of IAA phytohormone at 10−4 molar level involved the highest values
between all concentrations that used for IAA as well.
El-Nemr et al. [56] reported similar results. Since plant growth is substantially de-
pendent on soil fertility parameters, it appears that the improved physical, chemical, and
biological characteristics of the cultivation bed with applied VC are the reasons behind the
improved plant growth rate compared to other treatments [57]. Additionally, the relative
advantage of VC can be attributed to increased plant growth due to growth stimulants
such as plant growth hormones, humic materials, microbial activity and biodiversity, and
improved soil fertility [5,58]. Research conducted to assess the effects of VC on the studied
plants and its comparison with the effect of CHA as well as HA mixed with plant growth
hormones and IAA suggest that VC had the best performance on the experimented plants
among other treatments. It was also observed that increased application of VC would
increase medical plant (Moringa oleifera) root growth and the number of fruits [59].

3.1.2. Leaf Area and SPAD Index

Results obtained from variance analysis of leaf area traits and SPAD index demon-
strated a significant difference between various treatment levels. The results of comparing
mean value with Duncan’s multiple range test at 5% probability level showed that VC
had a higher performance than CHA and IAA; notably, among VC treatments, 3% levels
VC-AS and VC-NSP treatments led to larger leaf area and higher SPAD index, respectively.
The largest leaf area with a value of 225.5 cm2 per pot was observed under 3% level of
VC-NSP. The CHA involved a higher value of leaf area index compared to IAA acid soil
application treatment with 34.64% increase (Figure 2a). Increasing the concentration and
consumption percentage or treatments increased the overall SPAD index; the highest SPAD
index value was observed as 46.43 cm2 per pot with VC-AS at 3% level. The lowest values
for these indices were observed at 0% level treatments (Figure 2b). Plant absorption and
assimilation are, to a considerable extent, controlled by the two main factors of leaf area
and photosynthesis per leaf area unit. Accordingly, increasing these two factors would
enhance plant growth and performance [60].
Improvements in plant growth conditions have been shown with treated enriched VC
application [51,52]. Mahanta et al. [61] reported that the chlorophyll content of leaves and
the activity of some enzymes in rice were significantly increased under the treatment of VC
enriched with Azotobacter and Azospirillum. Additionally, an increase in shoot growth and
yield of cowpea (Vigna unguiculata) has been reported following the addition of enriched
VC, and this increase was ascribed to the enhanced availability of nutrients of enriched
VC, which in turn increases leaf chlorophyll content [62]. However, in our study, we did
not observe a significant difference in chlorophyll content (highest level used) between the
enriched treatments. It can be concluded that enrichment with bacteria and three important
 and the activity of some enzymes in rice were significantly increased under the treatment
of VC enriched with Azotobacter and Azospirillum. Additionally, an increase in shoot
growth and yield of cowpea (Vigna unguiculata) has been reported following the addition
of enriched VC, and this increase was ascribed to the enhanced availability of nutrients of
Plants 2022, 11, 227 enriched VC, which in turn increases leaf chlorophyll content [62]. However, in our study, 9 of 19
we did not observe a significant difference in chlorophyll content (highest level used) be-
tween the enriched treatments. It can be concluded that enrichment with bacteria and
three important
nutrients such asnutrients
N, P, andsuch as both
S were N, P,able
andtoSincrease
were boththe able to increase
nutrients the nutrients
availability to plants
availability to plants and, therefore, there was no significant difference
and, therefore, there was no significant difference between them. between them.

250 a
(a) a a a b bc b c
200 a b
Leaf area (cm2 pot-1)

b c VC
c c c d VC-N
150 d d
a a a a a a VC-AS
VC-NSP
100
CHA
IAA
50

Plants 2022, 10,0x FOR PEER REVIEW 10 of 20

1 2 3 4
Treatment levels

(b)
Chlorophyll index (SPAD)

50
VC
a
46 a ab a a b ab ab VC-N
a a a b b c c
42 b ab c VC-AS
a VC-NSP
a a a a a
38
CHA
34 IAA
30
1 2 3 4
Treatment levels

Figure 2. Comparison of average soil application of experimental treatments on leaf area (a) and
Figure 2. Comparison of average soil application of experimental treatments on leaf area (a) and
chlorophyll index (SPAD) (b) at treatment levels (levels 1, 2, 3 and 4 for VC, VC-N, VC-AS and
chlorophyll index (SPAD) (b) at treatment levels (levels 1, 2, 3 and 4 for VC, VC-N, VC-AS and VC- −6
VC-NSP treatments
NSP treatments of 0,of1,0,21,and
2 and
3%3% VC,VC, 0, 200,
0, 200, 400400
andand600600
mgmg of CHA
of CHA perper
kg kg of soil
of soil andand 0, −610
0, 10 , 10−5,
− 5 − 4 ≤ 0.05
10
and 10and 10 molar
−4 molar IAA). Different
IAA). Different letters
letters in each in eachshow
figure figure show significant
significant at p ≤ 0.05atbyp Duncan
difference
difference
by Duncanrange
multiple multiple
test. range
VC, VC test. VC, VCenrichment;
without without enrichment;
VC-N, VCVC-N, VC with
enriched enriched with 1% nitrogen;
1% nitrogen; VC-NSP,
VC-NSP,
VC enrichedVC enriched with 1% nitrogen,
with 1% nitrogen, 1% sulfur,1% and sulfur, and 1% phosphorus;
1% phosphorus; VC-AS, VC VC-AS, VCwith
enriched enriched with
Azotobacter
chroococcum
Azotobacter (21Az) + Pseudomonas
chroococcum fluorescensfluorescens
(21Az) + Pseudomonas (Ps 59); CHA, Commercial
(Ps 59); humic acid;
CHA, Commercial IAA,acid;
humic Indole-3-
IAA,
acetic acid.
Indole-3-acetic acid.

Numerous studies
Numerous studies suggest
suggest that
thatseveral
severalcomplex
complexcompounds
compounds areare
formed
formedbetween
betweenhu-
humic materialsand
mic materials andmineral
mineralionsionsduring
duringthethehumification
humification process;
process; this, in turn,
turn, increases
increases
enzyme
enzyme stimulation, their impact
stimulation, their impacton onraising
raisingrespiration
respiration intensity,
intensity, photosynthesis,
photosynthesis, andand
nu-
nucleic
cleic acid metabolism. Additionally, quasi-hormonal activity of humic materials waswas
acid metabolism. Additionally, quasi-hormonal activity of humic materials also
also reported
reported [63–65].
[63–65]. Asciutto
Asciutto et al.et[66]
al. [66]
also also reported
reported increased
increased leaf leaf
areaarea in Impatiens
in Impatiens wal-
walleranaas a result
leranaas a result of of 100–75%
100–75% VCVC treatment
treatment application.
application. Berova
Berova andand Karanatsidis
Karanatsidis [67][67]
ob-
observed increased
served increased photosynthetic
photosynthetic pigments
pigments inin pepper
pepper leaves
leaves following
following the
the application
application of
of VC. Golchin et al. [68] reported that the leaf area index and chlorophyll
VC. Golchin et al. [68] reported that the leaf area index and chlorophyll contents of pista- contents of
pistachio leaves
chio leaves werewere higher
higher under under VC treatment
VC treatment compared
compared to other
to other treatments
treatments without
without VC.
VC. The highest rates were observed in 10% and 20% VC treatments,
The highest rates were observed in 10% and 20% VC treatments, which increased by about which increased
by about
60%. 60%. Nutritional
Nutritional elements usedelements used for chlorophyll
for chlorophyll production production such phosphorus,
such as nitrogen, as nitrogen,
phosphorus, potassium, manganese,
potassium, manganese, iron, and copperiron, and
are copper are easily accessible
easily accessible to plants
to plants under VCunder
treat-
VC treatment
ment [69]. [69].

3.1.3. Stomatal Conductance and Photosynthesis

The results of mean value comparisons showed significant difference between vari-
ous treatments. In addition, VC-AS and VC-NSP treatments involved higher stomatal con-
ductance and photosynthesis while IAA treatment yielded the lowest values of both indi-
Plants 2022, 11, 227 10 of 19

3.1.3. Stomatal Conductance and Photosynthesis

The results of mean value comparisons showed significant difference between various
treatments. In addition, VC-AS and VC-NSP treatments involved higher stomatal conduc-
tance and photosynthesis while IAA treatment yielded the lowest values of both indices.
The highest extents of stomatal conductance and photosynthesis were observed under the
3% VC-NSP treatment with values of 15.57 and 21.26 µM CO2 × m−2 × s−1 , respectively
(Figure 3a,b). Increasing the concentration and consumption percentage of treatments
raised the overall values of both indices. Photosynthesis in plants is influenced by both
internal and external factors as well as environmental conditions. The internal factors of a
given cultivar determine the potential photosynthesis capacity. One of the internal factors
affecting the extent of photosynthesis is the health of leaves and presence of sufficient
chlorophyll within them; succulence of leaves results in increased photosynthesis rates [70].
A variety of factors including accessibility to sufficient water supply, suitable humidity
conditions, absence of water stress, and high water potential of leaves affect the extent
Plants 2022, 10, x FOR PEER REVIEW 11 of 20
of photosynthesis, directly or indirectly [71]. There is a direct relation between stomatal
conductance and photosynthesis in which increasing the former would enhance the latter.
Parallel changes between photosynthesis
Parallel changes and stomatal
between photosynthesis andconductance demonstrate
stomatal conductance the fact
demonstrate the fact
that maintaining photosynthesis can be attributed
that maintaining photosynthesis can beto retaining
attributed stomatalstomatal
to retaining conductance [72].[72].
conductance

18
(a)
(micro mol CO2 × m-2 × sec-1)

VC
ab a
Stomatal conductance

16 b VC-N
ab a cd d
cd bc VC-AS
d
14 a bc a ab e VC-NSP
d cd e
a a a a a a CHA
12 IAA

10

8
1 2 3 4
Treatment levels

22 b a a VC
(b) c c
(micro mol CO2 × m-2× sec-1)

VC-N
20 d d
ef e VC-AS
f fg f f fg f VC-NSP
Photosynthesis

18 g fg
CHA
h h h h h h h IAA
16

14

12

10
1 2 3 4
Treatment levels

Figure 3. ComparisonFigure
of average soil application
3. Comparison of average of
soilexperimental treatments on
application of experimental stomatal
treatments on conductance
stomatal conduct-
ance (a) and photosynthesis (b) at treatment levels (levels 1, 2, 3 and 4 for VC, VC-N, VC-AS and
(a) and photosynthesis (b) at treatment levels (levels 1, 2, 3 and 4 for VC, VC-N, VC-AS and VC- −6
VC-NSP treatments of 0, 1, 2 and 3% VC, 0, 200, 400 and 600 mg of CHA per kg of soil and 0, 10 ,
NSP treatments of 0,101, 2 and −6
−5 and 10−43% VC,
molar 0, 200,
IAA). 400 letters
Different and 600 mgfigure
in each of CHA
show per kg of difference
significant soil and at0,p10
≤ 0.05, by
− 5 − 4
10 and 10 molar IAA). Different letters in each figure show significant difference at p ≤ 0.05VC-
Duncan multiple range test. VC, VC without enrichment; VC-N, VC enriched with 1% nitrogen;
NSP, VC enriched with 1% nitrogen, 1% sulfur, and 1% phosphorus; VC-AS, VC enriched with Azo-
by Duncan multiple range test. VC, VC without enrichment; VC-N, VC enriched with 1% nitrogen;
tobacter chroococcum (21Az) + Pseudomonas fluorescens (Ps 59); CHA, Commercial humic acid; IAA,
VC-NSP, VC enriched with 1% nitrogen,
Indole-3-acetic acid. 1% sulfur, and 1% phosphorus; VC-AS, VC enriched with
Azotobacter chroococcum (21Az) + Pseudomonas fluorescens (Ps 59); CHA, Commercial humic acid; IAA,
Indole-3-acetic acid. 3.2. Effect of Using Different Treatments on Dry and Wet Weights of Aerial Organ, Root, and
Aerial Organ to Root Ratio
The effect of different amendments applied at various quantity levels were found to
be significant at 1% probability level for the entire set of said traits. The results of the mean
value comparison showed that the highest values for wet and dry weights of the aerial
organ and root were obtained from applying the 3% VC treatment. The highest wet and
dry weight values of the aerial organ were 123.7 and 12.28 g, respectively (Figure 4a,d);
the wet and dry weights of the root were also found to be 9.98 and 2 g, respectively (Figure
4b,c). The lowest and highest values for the dry weights of aerial organ to root ratio were
Plants 2022, 11, 227 11 of 19

3.2. Effect of Using Different Treatments on Dry and Wet Weights of Aerial Organ, Root, and
Aerial Organ to Root Ratio
The effect of different amendments applied at various quantity levels were found to
be significant at 1% probability level for the entire set of said traits. The results of the mean
value comparison showed that the highest values for wet and dry weights of the aerial
organ and root were obtained from applying the 3% VC treatment. The highest wet and dry
weight values of the aerial organ were 123.7 and 12.28 g, respectively (Figure 4a,d); the wet
and dry weights of the root were also found to be 9.98 and 2 g, respectively (Figure 4b,c).
The lowest and highest values for the dry weights of aerial organ to root ratio were obtained
from the applications of IAA and VC-AS and VC-NSP treatments, respectively.
Plants 2022, 10, x FOR PEER REVIEW 12 ofA20positive
correlation was observed between shoot and root dry weight with increasing concentration
of treatments used. Asciutto et al. [66] observed that increasing the amount of VC (75–100%)
at the cultivation
of VC (75–100%)bed would
at the enhance
cultivation the dry
bed would weights
enhance the of
dryboth the of
weights aerial
both organ and root
the aerial
in Impatiens
organ andwalleranaas. Thewalleranaas.
root in Impatiens use of VCThe positively
use of VCaffects dryaffects
positively matter,dryseed performance,
matter, seed
performance,
protein contents,protein contents,
and plants’ and plants’
nutrient nutrient absorption.
absorption. Such aimpact
Such a positive positiveisimpact is
probably due
probably due to the higher amounts of nutritional elements which, in turn,
to the higher amounts of nutritional elements which, in turn, results in the availability results in the
availability
of macro and of macronutrients
micro and micro[73].
nutrients [73]. Chanda
Chanda et al.reported
et al. [57] [57] reported enhanced
enhanced per-
performance
formance as a result of increased VC amounts. Such an increase is speculated to be the
as a result of increased VC amounts. Such an increase is speculated to be the result of
result of higher amounts of accessible nitrogen which is necessary for the production of
higher amounts of accessible nitrogen which is necessary for the production of structural
structural proteins. Compost and VC contain large amounts of humic materials (About 5
proteins. Compost
to 15 percent haveand VCreported
been containinlarge amounts
various ofinhumic
studies) materials
addition (About
to nutrients and 5organic
to 15 percent
have been reported
materials; in various
by enhancing studies) in of
the bioavailability addition to nutrients,
particular nutrientsesp.andiron
organic materials;
and zinc [74] by
enhancing the bioavailability
and directly of particular
affecting plant metabolism [75]nutrients, esp. increase
these materials iron andplant
zincgrowth
[74] and anddirectly
affecting plant metabolism [75] these materials increase plant growth and performance.
performance.

160
(a) VC
Shoot fresh weight (g pot-1)

a VC-N
120 a b b
c c VC-AS
a a d d
b c d d VC-NSP
d e e CHA
80
e
a a a a a a IAA

40

0
1 2 3 4
Treatment levels

12 VC
10
(b) a VC-N
Root fresh weight (g pot-1)

VC-AS
a a b
b c VC-NSP
8 a a d e
b c c CHA
c
6 d d f IAA
e
a a a a a a
4

0
1 2 3 4
Treatment levels

Figure 4. Cont.
Plants 2022, 11, 227
Plants 2022, 10, x FOR PEER REVIEW 13 of 20
12 of 19

2.5
a
VC
(c) a a b
Root dry weight (g pot-1)

2 b
ab a b VC-N
b c c
c c c VC-AS
1.5 d d d
VC-NSP
e
a a a a a a CHA
1
IAA
0.5

0
1 2 3 4
Treatment levels

15
VC
(d) a
Shoot dry weight (g pot-1)

12 a b c b VC-N
a c d VC-AS
c c b c c d
9 d VC-NSP
d e
e CHA
a a a a a a
6 IAA

0
1 2 3 4
Treatment levels

Figure 4. Comparison
Figure 4. Comparisonofofaverage
averagesoil
soil application
application ofofexperimental
experimentaltreatments
treatments onon shoot
shoot fresh
fresh weight (a),
weight
(a), Root fresh weight (b), root dry weight (c) and shoot dry weight (d) at treatment
Root fresh weight (b), root dry weight (c) and shoot dry weight (d) at treatment levels (levels levels (levels 1, 1,
2, 3 and 4 for VC, VC-N, VC-AS and VC-NSP treatments of 0, 1, 2 and 3% VC,
2, 3 and 4 for VC, VC-N, VC-AS and VC-NSP treatments of 0, 1, 2 and 3% VC, 0, 200, 400 and 0, 200, 400 and 600
mg of CHA per kg of soil and 0, 10−6, 10−5 and 10−4 molar IAA). Different letters in each figure show
600 mg of CHA per kg of soil and 0, 10−6 , 10−5 and 10−4 molar IAA). Different letters in each figure
significant difference at p ≤ 0.05 by Duncan multiple range test. VC, VC without enrichment; VC-N,
show
VCsignificant difference
enriched with at p ≤
1% nitrogen; 0.05 byVC
VC-NSP, Duncan multiple
enriched with 1%range test.1%
nitrogen, VC, VC without
sulfur, enrichment;
and 1% phos-
VC-N, VCVC-AS,
phorus; enriched
VC with 1% with
enriched nitrogen; VC-NSP,
Azotobacter VC enriched
chroococcum with 1% nitrogen,
(21Az) + Pseudomonas 1%
fluorescens (Pssulfur,
59); and
CHA, Commercial humic acid; IAA, Indole-3-acetic acid.
1% phosphorus; VC-AS, VC enriched with Azotobacter chroococcum (21Az) + Pseudomonas fluorescens
(Ps 59); CHA, Commercial humic acid; IAA, Indole-3-acetic acid.
3.3. Effect of Using Different Treatments on Seed Performance, Inflorescence Lengths, and Oil
3.3.Percentage in SeedsDifferent Treatments on Seed Performance, Inflorescence Lengths, and Oil
Effect of Using
Percentage
Thein Seeds comparison of mean values showed that the effect of the used treat-
statistical
ments
The as significant
statistical at 1% level.of
comparison Increasing the concentration
mean values showed thatand thepercentage of treatments
effect of the used treatments
as increased
significant theat
total
1%weight
level.ofIncreasing
seeds. Accordingly, VC-AS treatment
the concentration and at 3% level (2.55
percentage g per
of treatments
increased the total weight of seeds. Accordingly, VC-AS treatment at 3% levelin-(2.55 g
shrub) had the highest performance while the percentage of effective clusters were
creased as well (Figure 5a). Given quasi-hormonal traits and the stimulating impact of
per shrub) had the highest performance while the percentage of effective clusters were
HA, increasing the extent of photosynthesis and stomatal conductance in the aforemen-
increased as well (Figure 5a). Given quasi-hormonal traits and the stimulating impact of HA,
tioned treatments can be the main cause behind the increase in seed weight [76]. Inflo-
increasing
rescence the extent
length of photosynthesis
measurement and stomatal
results demonstrated thatconductance in thebelonged
the longest length aforementioned
to
treatments can be the main cause behind the increase in seed weight
VC-AS treatment (94.2 cm), while the shortest was observed under CHA and IAA [76]. Inflorescence
treat-
length
ments.measurement
These findingsresults demonstrated
were consistent with thethat theobtained
results longestfromlength belonged
the number to VC-AS
of clus-
treatment (94.2 cm), while the shortest was observed under CHA and IAA
ters and increased number of inflorescence length cluster showed a significant increase as treatments.
These
wellfindings were
(Figure 5b). consistent
Based with theresults,
on the obtained resultsitobtained from the
can be observed thatnumber of clusters
by increasing the and
load of treatments
increased number of enriched with N, P,
inflorescence and S,cluster
length a significant
showeddecrease from 3% toincrease
a significant 4% can be as well
(Figure 5b). Based on the obtained results, it can be observed that by increasing the load of
treatments enriched with N, P, and S, a significant decrease from 3% to 4% can be observed.
This decrease could be attributed to the very high sensitivity of plants in the flowering
stage, which increased or exceeded nutrients demand in this stage and can have the reverse
effect and reduce flowering, growth, and yield [77]. Furthermore, no significant effect was
Plants 2022, 10, x FOR PEER REVIEW 14 of 20

Plants 2022, 11, 227

observed. This decrease could be attributed to the very high sensitivity of plants in the
13 of 19
flowering stage, which increased or exceeded nutrients demand in this stage and can have
the reverse effect and reduce flowering, growth, and yield [77]. Furthermore, no signifi-
cant effect was observed between the treatments under measurement in the analysis of oil
observed between the treatments under measurement in the analysis of oil percentage
percentage value (Figure 6). The total amount of oil in rapeseed was reported as 40–45%
value (Figure 6). The total amount of oil in rapeseed was reported as 40–45% [78] which
[78] which was inconsistent with the results of this study. Certain reasons behind such
was inconsistent with the results of this study. Certain reasons behind such shortage of oil
shortage of oil in rapeseed compared to previous reports can be greenhouse cultivation
in rapeseed compared to previous reports can be greenhouse cultivation conditions and the
conditions and the
lack of fertilizer lackduring
supply of fertilizer supply
the growth during
period. the growth
Similarly, period. Similarly,
Balachandar Balachan-
et al. [51] reported
dar et al. [51] reported that bacterial-enriched VC had the greatest improvement
that bacterial-enriched VC had the greatest improvement in plant growth, grain yield, inand
plant
growth, grain yield,
leaf chlorophyll and The
content. leafnutrient
chlorophyll content.
content of theThe nutrient
plants as wellcontent of the plants as
as VC supplemented
well as VC supplemented with C, N, P, and K were significantly
with C, N, P, and K were significantly increased by enrichment. increased by enrichment.

3
a
(a) a VC
VC-N
Grain yield (g pot-1)

2 a VC-AS
b
VC-NSP
CHA
b c c
1 c c IAA
c d
b
c c c c d
a a a a a a e
0
1 2 3 4
Treatment levels

200 a
(b) a
VC
Inflorescence length (cm)

150 VC-N
VC-AS
a VC-NSP
100 ab
b CHA
IAA
b b b
50 b b c c c
a a a a a a b b b b b

0
1 2 3 4
Treatment levels
Figure 5.
Figure Comparison of
5. Comparison of average
averagesoilsoilapplication
applicationofofexperimental
experimentaltreatments
treatments ononGrain
Grainyield (a)(a)
yield and and
Inflorescence length
Inflorescence length (b)
(b) at
at treatment
treatment levels
levels(levels
(levels1,1,2,2,3 3and
and4 4forfor VC,
VC, VC-N,
VC-N, VC-AS
VC-AS andand VC-
VC-NSP
NSP treatments
treatments of20,and
of 0, 1, 1, 23%
andVC,3%0,VC,200,0,400
200,and
400600
andmg 600ofmg CHAof CHA
per kgper of kg
soilofand
soil0,and
10−60, 10−5−and
, 10 6,

10 −5molar
10−4 and 10 −4 molar
IAA). Different
IAA).letters in each
Different figure
letters show
in each significant
figure differencedifference
show significant at p ≤ 0.05 p ≤Duncan
at by 0.05
multiple range test. VC, VC without enrichment; VC-N, VC enriched with
by Duncan multiple range test. VC, VC without enrichment; VC-N, VC enriched with 1% nitrogen; 1% nitrogen; VC-NSP,
VC enriched
VC-NSP, VC with 1% nitrogen,
enriched 1% sulfur,1%
with 1% nitrogen, and 1% phosphorus;
sulfur, VC-AS, VC
and 1% phosphorus; enriched
VC-AS, with Azotobacter
VC enriched with
chroococcum (21Az) + Pseudomonas fluorescens (Ps 59); CHA, Commercial
Azotobacter chroococcum (21Az) + Pseudomonas fluorescens (Ps 59); CHA, Commercial humic humic acid; IAA, Indole-3-
acid; IAA,
acetic acid.
Indole-3-acetic acid.
Plants 2022,
Plants 2022, 11,
10, 227
x FOR PEER REVIEW 1514of
of 20
19

40 a a a
a a a

30
Seed oils (%)

20

10

0
VC VC-N VC-AS VC-NSP CHA IAA

Treatments
Figure
Figure 6.
6. Comparison
Comparison of of average
average soil
soil application
application of
of experimental
experimental treatments
treatments onon seed
seed oils
oils at
at different
different
treatments.
treatments. Different letters in each figure show significant difference at p ≤ 0.05 by Duncan multiple
Different letters in each figure show significant difference at p ≤ 0.05 by Duncan multiple
range
range test.
test. VC,
VC, VC
VC without
without enrichment;
enrichment; VC-N,
VC-N, VCVC enriched
enriched with
with 1%
1%nitrogen;
nitrogen; VC-NSP,
VC-NSP, VCVC enriched
enriched
with 1% nitrogen, 1% sulfur, and 1% phosphorus; VC-AS, VC enriched with Azotobacter chroococcum
with 1% nitrogen, 1% sulfur, and 1% phosphorus; VC-AS, VC enriched with Azotobacter chroococcum
(21Az) + Pseudomonas fluorescens (Ps 59); CHA, Commercial humic acid; IAA, Indole-3-acetic acid.
(21Az) + Pseudomonas fluorescens (Ps 59); CHA, Commercial humic acid; IAA, Indole-3-acetic acid.
3.4.
3.4. Effect
Effect of
of Using
Using Different
Different Treatments on Nutrients
Treatments on Nutrients Concentrations
Concentrations in
in the
the Aerial
Aerial Organ
Organ of
Rapeseed
of Rapeseed
3.4.1. Phosphorus
3.4.1. Phosphorus
The results
The resultsofof
variance
varianceanalysis of phosphorus
analysis contentcontent
of phosphorus measured in rapeseed
measured branches
in rapeseed
at the endatofthe
branches harvest
end ofshowed
harvestashowed
significant statisticalstatistical
a significant differencedifference
between between
various levels
variousof
treatments and different treatments. The highest and lowest amounts of
levels of treatments and different treatments. The highest and lowest amounts of phos- phosphorus were
obtainedwere
phorus fromobtained
the applications
from the of 3% VC-NSP
applications of (0.45%)
3% VC-NSP and indole-3-acetic acid, respec-
(0.45%) and indole-3-acetic
tively (Figure 7). Increasing the concentration and consumption percentage
acid, respectively (Figure 7). Increasing the concentration and consumption percentage of treatments of
increased the amounts of phosphorus in treatments. Ebrahimi
treatments increased the amounts of phosphorus in treatments. Ebrahimi et al.et al. [79] showed that[79]
as
an organic
showed thatsource, VC enhances
as an organic access
source, to nutrients
VC enhances including
access phosphorus,
to nutrients includingpotassium,
phosphorus,and
iron. Vermicompost
potassium, and iron. enhances phosphorus
Vermicompost enhances absorption
phosphorusby increasing
absorptionphosphorus
by increasing through
phos-
activating microorganisms via secreting organic acids such as citric, glutamic,
phorus through activating microorganisms via secreting organic acids such as citric, glu- succinic,
lactic, oxalic,
tamic, malic,
succinic, and
lactic, fumaric
oxalic, acids
malic, or stimulating
and fumaric acids phosphatase activity
or stimulating [80]. Considering
phosphatase activity
their high phosphorus contents, VC-NSP and VC-AS are probably the causes behind the
[80]. Considering their high phosphorus contents, VC-NSP and VC-AS are probably the
highest amounts of phosphorus witnessed in the plants placed under these treatments.
causes behind the highest amounts of phosphorus witnessed in the plants placed under
these
3.4.2. treatments.
Copper
The results of variance analysis on copper showed a significant difference between
0.5 the various levels of treatments. The highest and lowest amounts a of copper were observed
b
under VC and the CHA treatments (Figure a 8). Increasing the concentration and consump-
0.4 a
tion percentage of treatments b the amounts ofc copper in treatments.
increased VCIt is reported
P-concentration (%)

that HA createsbclow-soluble complexes

c d
with copper, which d
leads to reduced
VC-N uptake by
c cd d
ditsecontent was d e the measurement
0.3 plants [81]. Therefore, monitorede in canola shoot. Given
a a a a a a VC-AS
of the total copper contents of the plant, the reason for the increased amounts of copper
VC-NSP
0.2 in VC treatments can be attributed to the high extents of growth and dry weights if these
treatments. Moreover, the addition of VC bio-fertilizers would enhance CHA
access to and
IAA of the soil
absorption of trace elements such as copper by increasing the organic materials
0.1
and subsequently, increasing cation exchange capacity [82].
0
1 2 3 4
Treatment levels
 showed that as an organic source, VC enhances access to nutrients including phosphorus,
potassium, and iron. Vermicompost enhances phosphorus absorption by increasing phos-
phorus through activating microorganisms via secreting organic acids such as citric, glu-
tamic, succinic, lactic, oxalic, malic, and fumaric acids or stimulating phosphatase activity
[80]. Considering their high phosphorus contents, VC-NSP and VC-AS are probably the
Plants 2022, 11, 227 causes behind the highest amounts of phosphorus witnessed in the plants placed under 15 of 19

Plants 2022, 10, x FOR PEER REVIEWthese treatments. 16 of 20

0.5 a
b
Figure 7. Comparison of average soil applicationa of experimental treatments on P-concentration at
0.4 a
treatment levels (levels 1, 2, 3 and 4 for b
VC, VC-N, VC-AS and c VC-NSP treatmentsVCof 0, 1, 2 and 3%
P-concentration (%)

VC, 0, 200, 400 andbc 600 mg of CHAc per kg of soil and 0, 10−6,d10−5 and d10−4 molar IAA). Different
VC-N
c cd d significant d dat p ≤ 0.05 by Duncan multiple
e
0.3 letters in each figure show e difference e range test. VC, VC
a a a a without
a a
enrichment; VC-N, VC enriched with 1% nitrogen; VC-NSP, VC enriched with VC-AS1% nitrogen,
1% sulfur, and 1% phosphorus; VC-AS, VC enriched with Azotobacter chroococcumVC-NSP (21Az) + Pseudo-
0.2
monas fluorescens (Ps 59); CHA, Commercial humic acid; IAA, Indole-3-acetic acid.CHA
IAA
0.1 3.4.2. Copper
The results of variance analysis on copper showed a significant difference between
0 the various levels of treatments. The highest and lowest amounts of copper were observed
1 2 3 4
under VC and the CHA treatments (Figure 8). Increasing the concentration and consump-
Treatment levels
tion percentage of treatments increased the amounts of copper in treatments. It is reported
that HA creates low-soluble complexes with copper, which leads to reduced uptake by
Figure 7. Comparison of average soil application of experimental treatments on P-concentration at
plants [81]. Therefore, its content was monitored in canola shoot. Given the measurement
treatment levels (levels 1, 2, 3 and 4 for VC, VC-N, VC-AS and VC-NSP treatments of 0, 1, 2 and
of the total copper contents of the plant, the reason for the increased amounts of copper
3% VC, 0, 200, 400 and 600 mg of CHA per kg of soil and 0, 10−6 , 10−5 and 10−4 molar IAA).
in VC treatments can be attributed to the high extents of growth and dry weights if these
Different letters in each figure show significant difference at p ≤ 0.05 by Duncan multiple range
treatments. Moreover, the addition of VC bio-fertilizers would enhance access to and ab-
test. VC, VC without enrichment; VC-N, VC enriched with 1% nitrogen; VC-NSP, VC enriched with
sorption of trace elements such as copper by increasing the organic materials of the soil
1% nitrogen, 1% sulfur, and 1% phosphorus; VC-AS, VC enriched with Azotobacter chroococcum (21Az)
and subsequently, increasing cation exchange capacity [82].
+ Pseudomonas fluorescens (Ps 59); CHA, Commercial humic acid; IAA, Indole-3-acetic acid.

25

a a a VC
20 a a ab a a
b
Cu concentration (mg/kg)

b ab b VC-N
a a a a a a c c
c c d d
VC-AS
15
VC-NSP
CHA
10
IAA
5

0
1 2 3 4
Treatment levels
Figure 8.
Figure Comparison of
8. Comparison of average
average soilsoil application
application of
of experimental
experimental treatments
treatments on on P-concentration
P-concentration at at
treatmentlevels
treatment levels(levels
(levels1,1,2,2,3 3and
and4 for
4 for VC,
VC, VC-N,
VC-N, VC-AS
VC-AS andand VC-NSP
VC-NSP treatments
treatments of 0,of1,0,2 1,
and 2 and
3%
VC, 0, 200, 400 and −5 − 6 , 10 −−4
5 molar −4 molar
3% VC, 0, 200, 400 600
andmg 600ofmg CHA per kgper
of CHA of kg
soilofand 10−6,0,1010
soil0,and and 10 and 10IAA). Different
IAA).
letters in each figure show significant difference at p ≤ 0.05 by Duncan multiple
Different letters in each figure show significant difference at p ≤ 0.05 by Duncan multiple range range test. VC, VC
without enrichment; VC-N, VC enriched with 1% nitrogen; VC-NSP, VC
test. VC, VC without enrichment; VC-N, VC enriched with 1% nitrogen; VC-NSP, VC enriched withenriched with 1% nitrogen,
1%
1% sulfur,
nitrogen,and
1%1% phosphorus;
sulfur, VC-AS, VC VC-AS,
and 1% phosphorus; enrichedVC with Azotobacter
enriched chroococcumchroococcum
with Azotobacter (21Az) + Pseudo-(21Az)
monas fluorescens (Ps 59); CHA, Commercial humic acid; IAA, Indole-3-acetic acid.
+ Pseudomonas fluorescens (Ps 59); CHA, Commercial humic acid; IAA, Indole-3-acetic acid.

4.
4. Conclusions
Conclusions
The
The role
role of
of vermicompost
vermicompost (VC)(VC) enrichment
enrichment treatments
treatments isis effective
effective in
in increasing
increasing the
the
growth
growth andand yield
yield of
of canola.
canola. According
According to to the
the obtained
obtained results,
results, the
the added
added of of VC
VC atat the
the
highest
highest application levels
levels yielded
yieldedthe
thebest
bestresult,
result,while
whilethe
theindole-3-acetic
indole-3-aceticacid
acid was
was found
found to
to be the least stimulating amendment that showed the least positive impacts
be the least stimulating amendment that showed the least positive impacts in the measured in the meas-
ured indices.
indices. Vermicomposts
Vermicomposts NSP (enriched
NSP (enriched with nitrogen
with nitrogen (urea),(urea),
sulfur, sulfur, and phospho-
and phosphorus) and
AS (bioaugmented
rus) with Azotobacter
and AS (bioaugmented chroococcum
with Azotobacter and Pseudomonas
chroococcum fluorescens) fluorescens)
and Pseudomonas treatments
involved a involved
treatments significanta and higher and
significant effect on values
higher effectofon
morpho-physiological indices suchin-
values of morpho-physiological as
height,
dices dryas
such weight of dry
height, aerial and root
weight organs,
of aerial andSPAD
rootindex, and
organs, seed index,
SPAD performance,
and seed compared
perfor-
mance, compared to other treatments. The VCs NSP and AS treatments involved larger
amounts of seeds compared to other treatments. The type of elements which was used for
Plants 2022, 11, 227 16 of 19

to other treatments. The VCs NSP and AS treatments involved larger amounts of seeds
compared to other treatments. The type of elements which was used for enrichment was
very important. The results in this experiment showed that enrichment of VC with urea is
the weakest type of enrichment between all elements used. Therefore, for the enrichment
of VC, it seems that other enrichment treatments such as ammonium sulfate, etc. should
be studied.

Author Contributions: Conducted experimental verification, analyzed the data, A.H.; Contributed to
the research idea and designed the study, H.A.A.; draft preparation writing—original article, L.A. and
M.B.; Helped with constructive discussions and revised the article, B.A.L. and E.D.v.H. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data, tables, figures and results in paper are our own and original.
Conflicts of Interest: The authors declare that they have no conflicts of interest.

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