Scientia Horticulturae 261 (2020) 109015

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

The effect of chitosan, arbuscular mycorrhizal fungi, and compost applied T

individually or in combination on growth, nutrient uptake, and stem
anatomy of tomato
Fatima El Ameranya,b,c,*, Mohammed Rhazia, Said Wahbic, Moha Taourirteb, Abdelilah Meddichc
a
Natural Macromolecules Team, Normal Graduate School, Department of Biology, Cadi Ayyad University, Marrakech, 40000, Morocco
b
Laboratory of Bio-Organic Chemistry and Macromolecular, Faculty of Science and Technology of Marrakech, Department of Chemistry, Cadi Ayyad University,
Marrakech, 40000, Morocco
c
Laboratory of Biotechnology and Plant Physiology, Faculty of Science Semlalia Marrakech, Department of Biology, Cadi Ayyad University, Marrakech, 40000, Morocco

A R T I C LE I N FO A B S T R A C T

Keywords: This study investigated the effect of chitosan and a complex of arbuscular mycorrhizal fungi (AMF) on tomato
Nutrient deficiency plant (Solanum lycopersicum) grown in soil with four doses of compost (C0%, C5%, C10%, and C20%).
Bio-fertilizers Scanning electron microscopy with energy dispersive x-ray analysis revealed the interaction of NH2 groups of
Soil amendment chitosan with compost nitrogen that was increased by 80% after 24 h of cross-linking. Growth and physiological
Plant nutrition
parameters (root length, fresh and dry biomass of shoot and root, leaf area and Fv/Fm) and biochemical
Solanum lycopersicum
parameters (sugar and protein content) were improved in mycorrhizal plants grown in soil amended with
chitosan and 10% of compost (Ch+C10%M+) compared to control plants (ControlC0%M−). This improvement
was correlated with the increase of the diameter and the number of xylem vessels of tomato stems. In addition,
the application of chitosan and AMF together on plants grown in a poor soil of mineral elements (Ch+C0%M+),
increases the stem cortex that is involved in the distribution of minerals.
Chitosan application with AMF and 10% of compost was found to be the best treatment for tomato growth.
Therefore, adjusting soil with these three bio-fertilizers could be an interesting agricultural tool to help plants
adapt in poor soil.

1. Introduction physiological functions as reduction of growth and leaf senescence. On

The growth of a plant could be influenced by several environmental
factors, such as biotic and abiotic stress that damage the physiological
characteristics of plants (Atkinson and Urwin, 2012). Biotic stress is due
to living organisms destroying or attacking plants. While, abiotic stress
is triggered by changing the conditions of an especial environment that
is compulsory for normal development of plants such as water deficit,
cold or high temperature, salinity, mineral toxicity, and mineral defi-
ciency of soil and low or high light (Cramer et al., 2011). However,
some plant species are able to continue growing under stress conditions.
The responses of plants depend on the type, the duration of stress, and
the tissue affected by stress (Cramer et al., 2011). For example, the
decrease of available mineral elements in soil is a serious problem
which can lead to physiological disorders of plants. The mineral need is
differentiated from one plant to another. In addition, there are some
plants that require a high-level of nutrients in the soil. Consequently in
the case of deficiency, plants show responses to perturbation of
the other hand, there are also plants that have adapted to low-level of
nutrients in the soil. These plants have a high capacity to acquire mo-
bile nutrients such as potassium and a low capacity to get less mobile
nutrients such as phosphate (Chapin, 1987).
Tomato plant is very sensitive to nutrient deficiency (Gerloff, 1987).
Thus, it is important to provide enough water as well as mineral ele-
ments whether by a nutrient solution or by fertilizers.
Due to the decrease in the content of organic matter and native
fertility of the soil around the world, international attention focused on
adopting appropriate and sustainable strategies such as using bio-or-
ganic fertilizers from animal and vegetable waste or integrating mi-
croorganisms (mycorrhizal fungi), to make up for the lack of nutrients
and its effect on plant growth and to reduce the use of chemical ferti-
lizers.
Morocco generates a large amount of shellfish waste that is rejected
every day by restaurants or fishmongers. The valorization of shellfish
waste is a modern strategy; it reduces pollution and makes the waste

⁎
Corresponding author.
E-mail addresses: fatima.elamerany@ced.uca.ma, el.amerany.fatima@gmail.com (F. El Amerany).

https://doi.org/10.1016/j.scienta.2019.109015
Received 23 May 2019; Received in revised form 6 November 2019; Accepted 7 November 2019
Available online 19 November 2019
0304-4238/ © 2019 Elsevier B.V. All rights reserved.
F. El Amerany, et al.
useful in many fields. This strategy received a great deal of interest after
the discovery of chitin in the exoskeleton of crustacean shrimps and
crabs (Minke and Blackwell, 1978) that usually varied between 14 and
30 % of total waste materials. Recently, many derivatives of chitin are
prepared. The most used derivative in agriculture is chitosan that is
obtained from the deacetylation of chitin (Kumar, 2000). The beneficial
effects of chitosan on promoting plant growth and increasing biotic and
abiotic stresses tolerance have been reported (Pichyangkuraa and
Chadchawan, 2015). Furthermore, chitosan applied to soil as a plant
nutrient has shown a great effect on plant growth (Xu and Mou, 2018)
due to its improvement of soil fertility, and to its enhancement of mi-
neral nutrients absorption by plant (Berger et al., 2013). But, basic
research on biochemical, physiological and anatomical changes oc-
curred in plants under chitosan application is scarce and needs further
investigation.
The amendment of soil with bio-organic compounds such as com-
post was among the most successful strategies that still apply today.
Compost proved to promote growth because it’s rich in diversified de-
gradation products (Keeling et al., 1995), and to improve soil structure
(Ouédraogo et al., 2001). A previous study showed that the application
of compost had increased the tolerance of nutrient deficiency stress
(Walker and Bernal, 2008). But, no study is available on how the
amendment of soil with chitosan and compost would act on plant
growth and interact between them. We have expected that the combi-
nation of chitosan with compost, the organic amendment that is able to
bring more mineral elements, could be useful for promoting plant
growth.
Otherwise, the application of the symbiotic organisms as arbuscular
mycorrhizal fungi (AMF) has a positive effect on plant growth and yield
(Rooney et al., 2009; Bowles et al., 2016) and increases the tolerance of
nutrient deficiency stress (Garcia and Zimmermann, 2014). AMF colo-
nization increases plant growth, especially in stressful conditions
(Nadeem et al., 2014). The formation of fine hyphal networks in colo-
nized roots not only improve mineral and water uptake (Rouphael
et al., 2015) but also maintain the air-filled porosity of soil (Bitterlich
et al., 2018).
The effect of AMF varies among plants and depends on soil (as pH)
and fungal species (Koomen et al., 1987). Cosme et al. (2018) showed
that more than 71% of all vascular plant species, like tomato, are able to
make symbiosis with AMF. However, other studies also showed that
using a multiple mycorrhizal inocula had a positive effect on plant
growth compared to a single one (Koomen et al., 1987; Duponnois
et al., 2013; Chen et al., 2017). Therefore, we proposed to test the effect
of a complex of AMF on plant growth in order to maximize its gain and
to improve the benefits. The addition of chitosan or compost has been
reported to increase hyphal growth and sporulation of AMF (Gryndler
et al., 2003; Velásquez and Pirela, 2016).
However, the effects of bipartite as well as tripartite combination
compost, AMF, and chitosan have not been well documented and need
to be investigated further. In this context, our work will illustrate the
effects of AMF, chitosan, and compost from quack grass as a source of
nutrient on tomato plant, in order to find a balance between all these
bio-fertilizers, which could lead to better promotion of plant growth.
2. Materials and methods
2.1. Growth media
The growth substrate was a mixture of non-sterile compost of green
waste with sterile sand. The compost was produced over a period of
four months at a local source in Morocco, the nursery of the urban
municipality in Marrakech. The composition of compost was a mixture
of quack grass (Elymus repens) plants and leaf residues of others. The
compost was composed of 0.577% N, 0.057% P, 0.008% K, 0.015% Mg,
0.004% Fe, 0.199% Ca, 0.011% Na, 0.006% Se, 0.006% Si and 0.010%
Al and it had a pH of 8,98. However, the sand was composed of 0.022%
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Scientia Horticulturae 261 (2020) 109015
N, 0.001% P, 0.001% K, 0.006% Mg, 0.012% Fe, 0.010% Ca, 0.002%
Na, 0.002% Si and 0.010% Al and it had a pH 9.31. In order to de-
termine the best growth condition, the compost was applied at different
concentrations 0, 5, 10 and 20% of the final substrates.
2.2. Preparation of chitosan
Chitosan was produced by the deacetylation of chitin that was ex-
tracted from the shells of Parapenaeus longirostris based on the proce-
dure described by AL Sagheer et al. (2009). The deacetylation of chitin
was produced according to a similar previous procedure, but with some
modifications. One gram of chitin was stirred in strong sodium hydro-
xide (60%) during a day at room temperature then it was heated for
10 h at 110 °C. The resulting chitosan was filtered, washed, dried, and
then weighed.
Seven mg of chitosan powder was immersed in the solution of
compost (10%) for a period of 24 h. Then, the chitosan powder was
recovered and dried for further analysis.
2.3. Characterization of chitosan before and after cross-linking with
compost
2.3.1. Determination of the degree of acetylation
The degree of acetylation (DA) of chitosan was determined using
Fourier Transform Infrared (FTIR) spectroscopy (JASCO FT/IR-4600).
Lyophilized chitosan was mixed with KBr pellets. The FTIR spectrum
was obtained using specific parameters of the frequency
(400−4000 cm−1) and the resolution (4 cm−1). The DA% in three
samples of chitosan was calculated using the formula of Baxter et al.
(1992).
2.3.2. Inorganic composition of chitosan before and after cross-linking with
compost
The major inorganic element and its abundance in chitosan before
and after its association with compost were analyzed using scanning
electron microscopy (SEM)/ energy dispersive X-ray (EDX) analyzer
(TESCAN VEGA 3).
2.4. Plant growth studies
Disinfected seeds (Solanum lycopersicum L. cv. Campbell 33) were
germinated at 28 °C and then the seedlings were transplanted into pots
containing 2 kg of compost/sand mixture as previously indicated.
During transplanting, mycorrhizal plants were inoculated with 2.2 g of
arbuscular mycorrhizal fungi (AMF) complex, contained a mixture of
native species (2500 spores 100 g-1 sol: Glomus sp. (15 spores g-1 soil),
Sclerocystis sp. (9 spores g-1 soil), and Acaulospora sp. (1 spore g-1 soil)).
While, Non-AM plants received the same weight of autoclaved in-
oculums.
The experimental pots were placed in a greenhouse at 24 °C with
330 μmol.m-2.s-1 photosynthetic photon flux density and 69 % relative
humidity. After three weeks, chitosan was applied to the soil in the
transplant cavity at 1 mg/plant. The experiment was completely ran-
domized, with sixteen treatments crossing two mycorrhizal inoculation
levels (non-AM and AM) with four concentrations of compost (C0%,
C5%, C10% and C20%) and two chitosan levels (Control and Ch). Eight
replicates of each treatment were applied and the experimental unit
consisted of one pot containing seven tomato plants.
2.5. Plant analysis
All plant analysis was carried-out after 12 weeks of growth. The
shoot fresh weight (SFW) was measured after harvest. The roots were
extracted from the soil, washed, dried carefully with soft paper towel,
and used to determine root length (RL), then weighed to determine
their fresh weight (RFW). The leaves, the stems, and the roots were
F. El Amerany, et al.
placed in an oven at 70 °C for 48 h to determine their dry weight (SDW
and RDW). Biomass was determined by using three replicates per
treatment.
AMF colonization frequency and intensity were determined by
staining according to the method described by Phillips and Hayman
(1970), using fresh root and three replicates per treatment. In brief, fine
roots were cleared with 10% KOH at 90 °C for 15 min and washed three
times with distilled water. Subsequently, the roots were acidified with
1% (v/v) lactic acid at room temperature for 7 min. Then, the roots
were stained with 0.1% Trypan blue at 90 °C for 10 min. After staining,
roots were rinsed with water and stored at 4 °C for microscopic ob-
servation. 30 root segments with a length of 1–3 cm per plant are taken
and observed by using an optical microscopy. AMF colonization fre-
quency and intensity were calculated using the method described by
Trouvelot et al. (1986).
The efficiency of the photosystem II (Qy) was measured on healthy
leaves using chlorophyll fluorometer (Opti-sciences OSI 30p) after
20–30 min of dark adaptation. The relative fluorescence values of initial
(F0), maximal (Fm) and variable (Fv) fluorescence were assessed. The
PSII efficiency was expressed as the Fv/Fm ratio (Lutts et al., 1996).
Leaf area (LA) was determined by using scanner image leaf area
measurement Mesurim software.
In terms of sugar content, a sample of 0.1 g of frozen material of
leaves and roots was homogenized with 4 ml 80% (v/v) ethanol and
boiled in water bath in a 95 °C for three min. Then, the homogenate was
centrifuged at 2500 rpm for 5 min. The resulting supernatant was col-
lected, while the rest was suspended again by adding 2 ml of ethanol
and centrifuged. The first and the second supernatant were mixed and
used to quantify the total amount of sugar according to the method
described by Dubois et al. (1956), using glucose as a standard. Sugar
content was determined by using three replicates per treatment.
To determine protein content in leaves and roots, a sample of 0.25 g
of frozen materiel was homogenized with 0.5 ml of 0.02 M potassium
phosphate buffer, pH 7.6. Then, the homogenate was centrifuged at
10,000xg for 10 min at 4 °C. The supernatant was used to measure the
protein content according to the Bradford’s method (1976), using bo-
vine serum albumin as standard. The measurement of protein was done
using three replicates per treatment.
2.6. Determination of the mineral composition of tomato leaves
The amount of minerals in samples of tomato leaves was analysed
after 12 weeks of growth according to Segarra et al. (2007). A sample of
50 mg of dried leaves was digested with 1 ml HNO3, 1 ml deionized
water and 0.6 ml H2O2 in a teflon container at 90 °C for a period of 3
days. After digestion, the obtained solutions were diluted to 10 ml with
deionized water. The content of N, Ca, K, P, Mg, Se, Si, Cr, B, Cu, Fe,
Mn, Mo, Zn, and Na were determined by inductively coupled plasma-
atomic emission spectroscopy (ICP-AES). Three biological replicates
were performed.
2.7. Observation of stem anatomy
The stem anatomy of tomato was observed at the age of three
months, within a light microscope after preparation of cross-sections, in
similar distance positioned about 3 cm above the roots and stained
following the method according to Mondolot et al. (2001). Briefly, stem
cross-sections were immersed in a solution of sodium hypochlorite for
15 min, followed by three washing steps with water. They were then
immersed in 1% (v/v) acetic acid for 5 min and stained with Mirande’s
reagent for 15 min. The analysis of stems was carried out on eight re-
plicates per treatment using the VisiCam microscopy 2.1.
2.8. Statistical analysis
The statistical analysis was performed using CoStat 6.400 (CoHort
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Scientia Horticulturae 261 (2020) 109015
Table 1
Degree of acetylation (DA) of three samples of
chitosan.
Chitosan samples DA (%)
Sample 1 6.08
Sample 2 2.07
Sample 3 5.25
Software). Chitosan, AMF, compost levels and their interaction effect
were analyzed using factorial ANOVA. The comparison between mean
values in each factor was done using the Least Significant Difference
(LSD) test. Treatment effects were considered significant at P ≤ 0.05.
3. Results
3.1. Characterization of chitosan before and after cross-linking with
compost
3.1.1. FTIR spectroscopy analysis
The analysis of the IR spectrum (Table 1) of three samples of chit-
osan showed that the deacetylation step by 60% of NaOH allows re-
moving about 93.92% and 97.93% of the polymer acetyl unit.
3.1.2. EDX analysis
The objective of EDX spectrum analysis was to investigate the type
of element and its weight in percentage (Table 2). The EDX of chitosan
showed the presence only of carbon, oxygen, and nitrogen. Carbon and
oxygen were abundant in chitosan samples. The abundance of these
three elements was changed in chitosan linked with compost. The
comparison between chitosan before and after its association with
compost, demonstrates that the percentage of nitrogen was found to be
higher by 80% in chitosan linked with compost than chitosan alone
(Table 2).
3.2. Plant biomass and mycorrhizal root colonization
3.2.1. Effect of chitosan, compost, and mycorrhizal inoculation on plant
growth and physiology
The microscopic observation of roots (Fig. 1) showed that all roots
were colonized by AMF. However, the colonization varied in function of
soil composition (i.e., the concentration of compost / the presence or
the absence of chitosan).
The amendment of soil with chitosan (Ch+) has no significant (P ≤
0.05) effect on AMF colonization intensity at 8 weeks of growth stage.
Its promising effect was observed on this parameter after 12 weeks of
growth (P < 0.001) (Fig. 1 and Table S1). However, high root colo-
nization was observed in plant growing in low nutrients soil (C0%)
followed by 5% (C5%) then 10% (C10%) and finally 20% (C20%) of
compost (Fig. 1). Thus; AMF colonization frequency and intensity were
affected negatively by the application of compost, they decreased lin-
early as compost concentration increased at 8 and 12 weeks of growth
(P < 0.001) (Fig. 1 and Table S1).
The effect of combined treatment of chitosan with compost on AMF
colonization frequency and intensity was also studied (Fig. 1 and Table
S1). After 8 weeks of culture, the effect of chitosan was very remarkable
in nutrient-poor soil (0%) as well as 5% contrasted with soil amended
Table 2
EDX data of chitosan before and after cross-linking with compost.
Weight % in chitosan Weight % in chitosan linked with compost
C 52.40 44.70
N 07.69 13.87
O 39.90 41.42
F. El Amerany, et al.
with 10% and 20% of compost which lead to the reduction of root
colonization (Fig. 1A and B). After 12 weeks of culture, AMF coloni-
zation frequency and intensity reached to higher values of 46.77% and
96.66% respectively in plant grown in soil amended by chitosan alone
(Ch+C0% M+) (P ≤ 0.05) (Fig. 1C and D).
Moreover, the overall biomass measured as RL, SFW, SDW, RFW,
and RDW was significantly induced under the application of separated
(Ch+; AMF+; C10%) or combined (chitosan x compost; AMF x compost;
chitosan x AMF x compost) bio-fertilizers (Fig. 2 and Table S1). In
contrast, application of “chitosan x AMF” had no significant effect on
RL and shoot biomass (Table S1). Furthermore, all these parameters
were significantly (P ≤ 0.05) enhanced in mycorrhizal plants treated
with chitosan and 10% of compost (Ch+C10%M+) (Fig. 2A, B, C, D and
E). While, the growth of tomato plants was arrested under the appli-
cation of 20% of compost combined or non-combined with AMF and
chitosan (Fig. 2).
3.2.2. Effect of chitosan, compost, and mycorrhizal inoculation on leaf
growth and chlorophyll fluorescence parameters
To get deeper insight about the effect of three bio-fertilizers sepa-
rated or combined on tomato growth, leaf area (LA) and photosynthesis
parameters as chlorophyll fluorescence (Fv/Fm) were analyzed. LA was
significantly (P < 0.05) increased by about 40%, 41% and 393% in
plants treated with Ch+, AMF+ and C10%, respectively by comparison
to control (Fig. 2F). The application of bipartite combination had shown
a significant (P < 0.001) induction in LA (Fig. 2F and Table S1). The
effect of chitosan on leaf area (LA) was very remarkable in combined
treatment with AMF and 10% of compost (Ch+C10%M+) (Fig. 2F). The
LA was increased up to 12 % and 30 %, respectively in Ch+C10%M+
and Ch+C5%M+ compared with plants grown in soil unamended by
chitosan (ControlC10%M+ and ControlC5%M+ respectively) (Fig. 2F).
Regarding photosynthesis parameter, chitosan application did not
affect significantly the level of the quantum yield Fv/Fm (Table S1).
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Scientia Horticulturae 261 (2020) 109015
Fig. 1. Effect of chitosan and compost on AMF
colonization frequency ((A) after eight weeks
and (C) twelve weeks of colonization) and AMF
colonization intensity ((B) after eight weeks
and (D) twelve weeks of colonization). Data
are represented as the mean ± SD (n = 3).
Values in each column followed by the same
letter are not significantly different at P ≤ 0.05
(LSD test).
The application of AMF and C10% increased, however the level of Fv/
Fm by about 6% and 17%, respectively, in comparison to non-treated
plants (Fig. 2G). The effect of chitosan on Fv/Fm ratio was significantly
enhanced with compost or AMF application (Fig. 2G). Moreover, a
significant increase of Fv/Fm was observed in mycorrhizal plant grown
in the soil amended by 10% of compost (ControlC10%M+ and
Ch+C10%M+) (Fig. 2G).
3.2.3. Effect of chitosan, compost, and mycorrhizal inoculation on sugar
and protein content of leaves and roots
The level of total protein and sugar (leaves + roots) after 12 weeks
of growth was higher with fertilizers (Ch+; AMF; C10%) than without.
The level of sugar was much higher in plants inoculated with AMF than
in plants grown in soil amended with Ch+ or C10%, with respective
increases by 48%, 38%, and 16% in comparison to non-treated plants.
While, the highest level of protein was found in plants inoculated with
AMF followed by plants grown in soil amended with C10% then Ch+,
with respective increases by 61%, 30%, and 27% in comparison to
control (Fig. 3).
Additionally, the total amount of protein and sugar increased sig-
nificantly when bio-fertilizers were applied together (chitosan x AMF;
chitosan x compost; AMF x compost; chitosan x AMF x compost). A
significant (P ≤ 0.05) increase of total sugar (259.13 mg/g FW) and
protein (14.42 mg/g FW) contents was found in inoculated plant
treated with chitosan and C10% (Ch+C10%M+) in comparison to
control plant (207.08 mg/g FW and 9.30 mg/g FW, respectively)
(ControlC10%M+). In combined treatment, the contents of protein and
sugar were higher in root than shoot (Fig. 3).
3.2.4. Mineral composition of tomato leaves
Our results showed that the application of chitosan alone (Ch+) had
a greater impact on macro and micronutrients of shoot (Table S1). The
amount of N, Ca, Mg, Si, Fe, Mn and Na was significantly (P < 0.001)
F. El Amerany, et al.
Fig. 2. Effect of chitosan, AMF, and compost on: (A) root length (RL); (B) shoot fresh weight (SFW); (C) shoot dry weight (SDW); (D) root fresh weight (RFW); (E) root
dry weight (RDW); (F) leaf area (LA); (G) Fv/Fm. Data are represented as the mean ± SD (n = 3). Values in each column followed by the same letter are not
significantly different at P ≤ 0.05 (LSD test).
improved by 29%, 157%, 138%, 104%, 144%, 72%, and 81%, re-
spectively (Fig. 4). Colonization with AMF increased the amount of B,
Zn, and Na by 130%, 136% and 36% respectively; but, it had a negative
impact on the level of N in tomato leaves (Fig. 4). Additionally, no
significant differences were seen at the level of K, P, Se, Cr, Cu and Mo
(Fig. 4 and Table S1).
Regarding the treatments with combination, chitosan application
with AMF (Ch+C10%M+) improved the mineral element content in
leaves (Fig. 4 and Table S1); but, it had a little impact compared to the
application of chitosan alone (Ch+C10%M−).
3.3. Anatomical features of stem
Data presented in Table S1 showed that the anatomical features of
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Scientia Horticulturae 261 (2020) 109015
stem, like stem diameter, cortex width and diameter of pith and xylem’s
vessels, were significantly improved under the application of either
chitosan, AMF, or 10% of compost.
The interaction between these bio-fertilizers had a significant (P ≤
0.05) effect on stem features (Fig. 5 and Table S1).
When chitosan and AMF were applied together, the stem diameter
increased in plants grown in soil amended with both concentrations of
compost (C0% or C10%). In non-inoculated plants, no significant dif-
ference was observed between chitosan-treated and control plants. It
was noted that stem diameter was greater in plants treated with C10%
compared to plants grown without compost (Fig. 5).
The measure of cortex width showed that there was no significant
difference between treatments: ControlC10%M−, ControlC10%M+ and
Ch+C0%M+, which means that the application of chitosan and AMF
F. El Amerany, et al.
Fig. 3. Effect of chitosan, AMF, and compost at the level of sugar (A and B) and
protein (C and D) content in shoots (A and C) and roots (B and D) after twelve
weeks of culture. Data are represented as the mean ± SD (n = 3). Values in
each column followed by the same letter are not significantly different at
P ≤ 0.05 (LSD test).
together in soil with lower nutrients, improves the cortex width that is
similar to those provided with a normal nutrition. On the other hand,
the non-mycorrhizal plant treated with chitosan and 10% of compost
(Ch+C10%M−) had significantly (P ≤ 0.05) higher cortex width
(Fig. 5).
Chitosan application increased the pith diameter with the exception
of non-innoculated plants and cultivated in 0% of compost (Ch+C0%
M−) which had a smaller diameter (Fig. 5).
Concerning the xylem’s vessel diameter, it was increased in my-
corrhizal plants grown without compost compared to non-mycorrhizal
plants. The xylem vessels diameter was greater in mycorrhizal plants
treated with chitosan. The number of vascular bundles shown on my-
corrhizal plants treated with chitosan and 10% of compost (Ch+C10%
M+) was higher compared to control plants (Figs. 5–7).
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Scientia Horticulturae 261 (2020) 109015
4. Discussion
The application of three bio-fertlizers, chitosan (Ch+) from crusta-
cean shrimps, compost from quackgrass, and a complex of arbuscular
mycorrhizal fungi (AMF), either single or combined, significantly pro-
moted the growth of tomato plants. The data revealed that there was a
promotion of root length, fresh and dry weight of shoot and root, and
leaf area under the application of either Ch+ or compost. When these
two bio-fertilizers were applied together the growth parameters tackle a
significant rise due to their providing a regular supply of mineral nu-
trient (Padilla et al., 2017; Xu and Mou, 2018). Among the four doses of
compost applied, plants grown in soil amended with C10% achieved
greater growth. While, the amendment of soil with a higher quantity of
compost (C20%) had lower productivity. Compost is known as an ex-
cellent bio-fertilizer due to its slow release of nutrients (Rady et al.,
2016). Therefore, there is no dose at which compost becomes toxic to
plants. In contrast, when plants get more compost the growth increased
(Kong et al., 2017). But, the inhibition of growth under the application
of C20% could be explained by the fact that the super uptake of some
mineral nutrient by plant could interrupt the absorption of others.
Previous studies have shown that the increase of N supply has a ne-
gative effect on plant physiology and metabolism because of disruption
of N metabolism and decrease of K+, Ca+, and Mg2+ uptake (Chang
et al., 2012). The excessive uptake of NH4+ could be done through
plant K+ transporters and channels. Thus, the competition during the
uptake would decrease the uptake of K+ (Hoopen et al., 2010). In ad-
dition, the excessive N application may increase the content of reactive
oxygen species (ROS) and lipid peroxidation due to inactivation of
antioxidant enzymes, superoxide dismutase and peroxidase, that are
involved in ROS detoxification. As a consequence of metabolic changes,
leaf area, leaf number, flowers and grain filling period affected, and leaf
senescence accelerated (Chang et al., 2012; Kong et al., 2017).
With regards to AMF colonization, the growth parameters of tomato
were significantly enhanced in mycorrhizal plants in comparison to
non-inoculated plants. Furthermore, microscopic observation of roots
showed that AMF colonization frequency was reached 86% indicating
that the use of a complex of AMF may help to promote growth of to-
mato. In addition, AMF colonization was changed in the presence of
other bio-fertilizers (Ch+ and compost). When Ch+ applied alone with
AMF, the frequency and intensity of AMF root colonization were sig-
nificantly increased to reach 47% and 97%, respectively; however,
chitosan did not show an enhancement of growth of mycorrhizal plants.
Thus, the amendment of soil with Ch+ affected positively AMF colo-
nization which has been attributed to increase length of extra-radical
mycelium and stimulate fungal spore formation (Gryndler et al., 2003;
Velásquez et al., 2016); but, it was not sufficient to increase the plant
growth and to make up for non-existent nutrients. In contrast, the
amendment of soil with C5% and C10% has improved root length,
shoot and root biomass, and leaf area of mycorrhizal plants; but, the
mycorrhizal colonization was inhibited. In the present study, the supply
soil with a medium (C5% and C10%) and a high (C20%) dose of
compost increased the availability of phosphorus in soil; wherefore, the
mycorrhizal pathway, which is stimulated better in limiting conditions
and soil poverty, was deactivated (Bücking et al., 2012). The combi-
nation of three bio-fertilizers gave a greater promotion of tomato
growth than those plants supplied with individual fertilizer or control
plants. The mycorrhization was, however, reduced even the adding of
Ch+ was not helping. This result is in contrast with Johansson (1994).
The beneficial effect of these bio-fertilizers on plant physiology is
not related only to a better acquisition of water and nutrients but also
an increase of photosynthesis and carbohydrates (Hussein et al., 2006;
Dzung et al., 2011; Mandal et al., 2013). Our findings demonstrated
that the application of AMF and compost (especially C10%), in-
dividually or combined, has increased the chlorophyll fluorescence (Fv/
Fm) and the level of carbohydrates (protein and sugar); however, Ch+
application has less effect. The mycorrhizal symbiosis is translated to a
F. El Amerany, et al.
Fig. 4. Mineral nutrient content of leaves in response to treatments. Data are represented as the mean ± SD (n = 3). Values in each column followed by the same
letter are not significantly different (LSD test).
mutually beneficial exchange for both fungi and root. So, in order that
the symbiotic relationship is in balance, the fungi can offer water and
mineral nutrients to plants that in return it could transfer between 4
and 20% of carbon from the photosynthate to the fungi (Bücking et al.,
2012). When plants store more water stomatal opening is stimulated
resulting in an increase of carbon assimilation and a higher chlorophyll
content and phostosynthetic rate (Zhu et al., 2017). Santana et al.
(2015) reported that the application of compost increases the photo-
chemical efficiency. The effect of compost is related to an increase of
the rate of nutrient in the soil which influenced chlorophyll content and
Fv/Fm ratio of many crops (Santana et al., 2015; Srinivasarao et al.,
2016).
Furthermore, Ch+ applied soil was reported to increase photo-
synthetic rate (Xu and Mou, 2018). Its effect could be related to its
7
Scientia Horticulturae 261 (2020) 109015
nitrogen providing and to its involvement in absorbing nutrients by
roots (Bélair and Tremblay, 1995). In the present investigation, it was
clear that even the application of Ch+ had a little impact on the
chlorophyll fluorescence and the biosynthesis of protein and sugar; but,
its application in combination with AMF+ and C10% was probably
considered healthy to plants due to the increase of Fv/Fm rate that
reach 0.866, and the level of sugar and protein.
Based on the overall of this data, it seems that the effect of Ch+ on
growth could be due to its involvement in AMF signaling or in assim-
ilation of compost minerals. The effect of Ch+ varied and depended on
average size, structure, molecular weight and the degree of acetylation
(DA), and besides, the type of species studied and the concentration
applied. FTIR analysis of three samples of Ch+ indicated that it had
2.07–6.08% of N-acetylglucosamine (GlcNAc) units in its backbone.
F. El Amerany, et al.
Fig. 5. Stem diameter (A), cortex width (B), pith diameter (C), and xylem’s
vessel diameter (D) in stem of tomato plant after twelve weeks of growth, in-
oculated with AMF and grown in 4 substrates: 0 and 10% of compost amended
or unamended with chitosan. Data are represented as the mean ± SD (n = 8).
Values in each column followed by the same letter are not significantly different
at P ≤ 0.05 (LSD test).
GlcNAc is the monomer of chitin polysacharide, the main component of
the fungal cell wall and the important signal in the induction of plant
defense and the establishment of symbiosis (Gust et al., 2012). The
symbiosis with AMF relies on signaling molecules exchanged between
the host plant and the AMF. Upon sensing root of the host plant, fungus
germinates, two acids secreted by root (2-hydroxy tetradecanoic acid
and fatty acid) induce lateral branches elongated of hyphae. When
hyphae is near to the root, it senses strigolactones and branches more
complexly to become very closer to root and produces Myc factors,
8
Scientia Horticulturae 261 (2020) 109015
chitin oligomers (tetramers and pentamers) and a mixture of sulfated
and non-sulfated simple lipooligosaccharides (LCOs) (Gutjahr, 2014;
Schmitz and Harrison, 2014). At the moment of recognition of GlcNAc
of Myc factors by plant plasma membrane-localized lysin motif (LysM)
proteins, root secrets cutin monomers to induce penetration of hypha
inside root epidermis and the formation of arbuscules (Gust et al., 2012;
Schmitz and Harrison, 2014; Nadal et al., 2017). Chitin oligomers from
AMF as a microbe-associated molecular pattern don’t elicit the immune
system of plant. The inhibition of immunity signals as well as the role of
these oligomers is still poorly understood. Maillet et al. (2011) reported
that plant could discriminate between symbionts and pathogens based
on the length of chitin oligomers. The activation of immune system
requires long-chain chitin oligomers than the short-chain oligomers
(such as CO5), which is important for symbiosis (Liu et al., 2012; Genre
et al., 2013).
The effect of Ch+ or related chitin oligomers on plant growth,
photosynthesis, and carbohydrates production remains higher, al-
though there is an inhibition of AM symbiosis during intensive phos-
phate fertilization, suggesting that the recognition of chitin oligomers,
liberated after degradation of Ch+ by chitinolytic bacteria of compost
(Beier and Bertilsson, 2013), could help to increase tomato growth.
Further work will be necessary to identify all roles of chitin short chain
signaling.
On the other hand, it was reported that Ch+ function increases with
the decrease of DA (Lizárraga-paulín et al., 2011). Our data showed that
Ch+ backbone contains more than 94% of glucosamine (Glc) mono-
mers, which means more free amino groups. In addition, the amino
group of Ch+ has the ability of chelating ions (Sobahi et al., 2014),
suggesting its contribution in the assimilation of compost minerals. Our
data revealed that Ch+ applied soil had a greater impact on the as-
similation of N, Ca, Mg, Si, Fe, Mn, and Na than the application of
AMF+ which affects positively only B, Zn, and Na. The cationic groups
of Ch+ (-NH3+) could bind anionic nutrient of soil and compost as
phosphorus (H2PO4-, HPO42-, PO4-3, PO3-3), nitrogen (NO3- and NO2-),
and silicium (SiO4-4) ions. The comparison of chemical composition of
Ch+ before and after cross-linking with compost using EDX analysis,
revealed that the amount of N was found to be higher by 80% in Ch+
linked with compost, suggesting the interaction of –NH2 groups of Ch+
with nitrogen ions presented in soil. Fatima et al. (2018) noted that
Ch+ mineralized and released nutrients in the soil to be absorbed by
plants. Therefore, Ch+ effect on tomato growth could be related to its
chelating of compost ions which will be liberated after mineralization
of Ch+ over a longer time period.
Plant growth represents the process by which the number and the
size of cells had increased (Saucedo and Edgar, 2002). Cells growth is
not only performing the increase in the number and size of leaves,
flowers and fruits, but also the increase in the length and width of roots
and stem. The indeterminate growth of stem provides support to aerial
part of plant and conducts the exchange of water, mineral salts, sig-
naling molecules, amino acids, and carbohydrates between aerial and
underground organs (Dickison, 2000). The mechanisms leading to the
changes in stem structure remain uninvestigated, but the few studies
available had found that supplying soil with mineral nutrition increased
the diameter of stem and xylem vessels for better assimilation of water
and nutrients (Zhuk, 2014). Microscopic observation of cross-section of
stems showed a responsive and an enhancement of stem diameter,
cortex width and diameter of pith and xylem’s vessels under the ap-
plication of individual or combined bio-fertilizers. The improvement of
stem diameter under the application of either Ch+, AMF, or compost
has been reported (Zahid et al., 2014; Haouvang et al., 2017; Yang
et al., 2018; Ravindran et al., 2019). Furthermore, our data revealed
that the application of combined bio-fertilizers increased parenchyma
cells of pith which supported stem by filling with solid materials (i.e.,
sugar) (Kong et al., 2013). The application of Ch+ and AMF together on
plants deficient in mineral elements (C0%) increases the cortex layer
that becomes similar to those grown with a complete nutrient supply.
F. El Amerany, et al. Scientia Horticulturae 261 (2020) 109015

Fig. 6. Surface appearance of stem’s cross section of tomato plant inoculated with AMF and grown for twelve weeks in four substrats: 0 and 10% of compost amended
or unamended with chitosan (x40). Abbreviations: Cor, cortex; P, pith; X, xylem.

9
F. El Amerany, et al. Scientia Horticulturae 261 (2020) 109015

Fig. 7. Surface appearance of stem’s cross section of tomato plant inoculated with AMF and grown for twelve weeks in four substrats: 0 and 10% of compost amended
or unamended with chitosan (x100). Abbreviations: Cor, cortex; P, pith; X, xylem; V, xylem vessel.

10
F. El Amerany, et al.
This layer serves in transportation of water or substances through its
cells and serves as a storage of the product of photosynthesis (starch)
(Dickison, 2000). Some studies have demonstrated the localization of P,
K, Fe, Ca and Cl ions with a high quantity in the cortex layer of Ber-
kheya coddii root inoculated with Rhizophagus intraradices compared to
non-inoculated roots (Orlowska et al., 2013); however, there is no study
on cortex layer changes under the application of either Ch+ or compost.
The enrichment of mineral elements and the products of photosynthesis
in the cortex layer as indicated above suggest that the cortex layer could
play an important role in binding, assimilation, transport, and storage
(Orlowska et al., 2013).
The diameter and the number of xylem’s vessels of stem were in-
creased in AM plants. Daft and Okusanya (1973) found similar result in
tomato plant inoculated with endogen AMF. However, high growth of
xylem vessels occurred in plant grown in soil with combined bio-ferti-
lizers, indicating the enhancement of mineral nutrition.
The improvement of xylem growth was most likely attributed to soil
nitrogen content released by either chitosan or compost (Yu et al.,
2019) and the accumulation of cytokinin hormone which is known to
play a crucial role in promoting cell division and organ formation
(Kieber and Schaller, 2018). The response of cytokinin occurs in pro-
cambial cells not only leads to generation of xylem and phloem tissues
but also promotes polar auxin transport to xylem for its development
(Benková et al., 2003; Schaller et al., 2014; Moreno-Piovano et al.,
2017; Kieber and Schaller, 2018). In the future, the measurement of
cytokinin and auxin hormone concentrations would probably clarify the
effect of bio-fertilizers application on plant growth.
5. Conclusion
The use of three bio-fertilizers chitosan, AMF, and compost was
proven to be very beneficial for the growth of tomato plants. In our
study, the application of chitosan and AMF together on plants grown in
a poor mineral soil promotes changes in all parameters studied. These
two bio-fertilizers provide the minerals needed by the plant. Hence,
there is an increase of root and shoot growth, chlorophyll fluorescence,
and a remarkable improvement of stem tissues. Furthermore, chitosan
combined with AMF increased the content of sugar and protein, which
was directly correlated to the leaf area and the chlorophyll fluores-
cence.
The application of the three bio-fertilizers together looks more ef-
ficient than being separated. Indeed, the interaction of C10%, chitosan,
and AMF has increased significantly physiological, biochemical, and
anatomical properties of the studied parameters.
Declaration of Competing Interest
None.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Acknowledgments
This work was supported by Moroccan National Center for Scientific
and Technical Research (33UCA2015). Authors wish to thank the
Center for Analysis and Characterization UCA Marrakech Morocco for
supporting this study.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.scienta.2019.109015.
11
Scientia Horticulturae 261 (2020) 109015
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