Carbohydrate Polymers 284 (2022) 119233

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Disease resistance and growth promotion activities of chitin/cellulose

nanofiber from spent mushroom substrate to plant
Hujun Li a, Saori Yoshida a, Naofumi Mitani a, Mayumi Egusa b, Momoko Takagi b,
Hironori Izawa a, c, Teruyuki Matsumoto b, d, Hironori Kaminaka b, Shinsuke Ifuku a, c, *
a
Department of Engineering, Graduate School of Sustainability Science, Tottori University, 4-101 Koyama-Minami, Tottori 680-8552, Japan
b
Faculty of Agriculture, Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan
c
Center for Research on Green Sustainable Chemistry, Tottori University, 4-101 11 Koyama-Minami, Tottori 680-8550, Japan
d
Marine Nano-fiber Co., Ltd., 4-101 Koyama-Minami, Tottori 680-8550, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Some studies have reported the method for treating the spent mushroom substrate (SMS). However, the effective
Cellulose nanofibers use as a functional raw material based on properties of SMS remains a formidable challenge. In this study, we
Chitin nanofibers investigated the usefulness of SMS in agriculture to develop a new method for treating and utilizing it. First, we
Spent mushroom substrate
attempted to isolate chitin/cellulose nanofiber complex (CCNFC) from SMS using chemical pretreatment and
Plant disease resistance
Plant growth promotion
mechanical fibrillation. The characterization results like SEM, FT-IR, and XRD showed that we successfully
Functional agricultural nanomaterial isolated the CCNFC from SMS. Second, we explored the biological activities of the CCNFC for its potential
application as a functional agricultural nanomaterial. CCNFC water dispersion with low concentration (0.1 and 1
mg/mL) exhibited significant plant disease resistance and plant growth promotion activities. Our results sug­
gested that SMS may provide a useful source of functional agricultural nanomaterial, which may contribute to
treating and applying it in agriculture.

1. Introduction
Chitin and cellulose are the two most abundant, inexhaustible, and
renewable natural resources for human beings. Chitin is widely present
in fungi and algae's cell wall, the shell of arthropods such as shrimp,
crabs, insects, and the shell and cartilage of shellfish mollusks (Ifuku &
Saimoto, 2012; Jones et al., 2020). As the most abundant natural
resource on earth, cellulose mainly exists in the plants such as wood,
cotton, hemp, and various kinds of straw plants (Mishra et al., 2018),
which can be synthesized hundreds of millions of tons through photo­
synthesis every year (Boex-Fontvieille et al., 2014). With the increase of
attention to environmental pollution globally, the research and appli­
cation on chitin and cellulose are attracting worldwide attention due to
their cheap, abundant, biodegradable, non-toxic, and biocompatible
properties (Seddiqi et al., 2021). In particular, the development and
research on chitin nanofibers and cellulose nanofibers from various
agricultural residues have been actively conducted in the world recently
(Alemdar & Sain, 2008; Ifuku & Saimoto, 2012; Gabriel et al., 2020).
The efficient application of unused biomass, including agricultural
waste, will help solve environmental problems such as global warming
and promote the development of agriculture, forestry, fisheries, and
emerging industries.
Spent mushroom substrate (SMS) is an agricultural by-product from
mushroom cultivation, mainly composed of sawdust and rice bran.
Generally, 5 kg of SMS will generate for harvesting 1 kg of mushrooms
(Medina et al., 2012). The SMS increase with the rise in consumption of
mushrooms. Therefore, dealing with such a large amount of SMS has
become an urgent issue for a mushroom producer. Most of the SMS is
treated as industrial waste, which needs a vast amount of disposal costs
and pollutes the environment. Several studies have shown that SMS can
be used as organic fertilizer and soil conditioners (Owaid et al., 2017).
However, only a tiny portion of the SMS was used as fertilizer due to the
lack of stability and maturity (Paula et al., 2017). Besides, several
studies have also reported the bioactive compounds extracted from the
SMS. Yoshida et al. prepared fermented SMS (Yoshida et al., 2017),
increasing egg production and decreasing the proportion of broken and
soft-shelled eggs. Ishihara et al. and Tanaka et al. found that the hot
water extracts from the SMS could inhibit the germination of conidia

* Corresponding author at: Center for Research on Green Sustainable Chemistry, Tottori University, 4-101 11 Koyama-Minami, Tottori 680-8550, Japan.
E-mail address: sifuku@tottori-u.ac.jp (S. Ifuku).

https://doi.org/10.1016/j.carbpol.2022.119233
Received 19 October 2021; Received in revised form 18 January 2022; Accepted 5 February 2022
Available online 8 February 2022
0144-8617/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
H. Li et al. Carbohydrate Polymers 284 (2022) 119233

and prevent osteoporosis, respectively (Ishihara et al., 2018; Tanaka

et al., 2019). These reports focused on the direct applications of the SMS Spent mushroom substrate
as fertilizers, soil conditioners, or the extraction of related bioactive
compounds. No studies have evaluated the applications of the SMS as a
functional agriculture nanomaterial resource.
Recently, Konno et al. isolated cellulose nanofibers from the SMS Removal of fat
using TEMPO-mediated oxidation and prepared cellulose nanofiber film Toluene/ethanol (2/1(v/v), 3 days)
(Konno et al., 2016). Their results reveal the potential of the SMS as a
raw material for the isolation of cellulose nanofiber. However, they only
discussed the cellulose in the SMS, and another ingredient in the SMS
chitin was not discussed. As well known, after the harvest of mushrooms, Removal of lignin
the mycelium of mushrooms will remain in the SMS. That is to say, the x4
SMS contains not only cellulose from sawdust but also chitin from
NaClO2, acetic acid (75 ºC, 1 h)
mycelium. Therefore, the SMS is a kind of agricultural by-product with a
unique characteristic that differs from other agricultural wastes. To
exploit the characteristics of SMS, we think it is necessary to incorporate
this unique point into the experimental design for practically and Removal of hemicellulose
rationally using the SMS. It would benefit from exploiting the feature
6 wt% KOH (r.t., overnight)
and finding a more suitable way to treat and utilize SMS effectively. In
addition, considering the importance of agriculture to human beings, we
believe that if the SMS can well apply to agricultural production, it will
effectively solve the problem of SMS treatment but also help to promote
the development of agriculture.
Chitin/cellulose complex
In this study, we attempt to convert the SMS into a form of nanofiber
water dispersion and apply it to agriculture, due to the nanofiber water
dispersion having many advantages such as easy to use, storage and
transport. Our purpose is to develop a new method for treating and
Grinder treatment
applying the SMS as a functional raw material. To this end, we first
tested whether the chitin/cellulose nanofiber complex (CCNFC) can be
separated from the SMS. Second, we verified the usefulness of the
CCNFC in agriculture, including plant disease resistance and plant Chitin/cellulose nanofiber
growth promotion activities, to explore the potential application of
CCNFC. complex
Our study suggests a new approach to treating SMS: converting it
into nanofibers and using them in agriculture in the form of water Fig. 1. Isolation procedures for chitin/cellulose nanofiber complex from spent
dispersion. Our results demonstrated that SMS has great potential to mushroom substrate.
produce nanofiber complex and revealed that CCNFC might be a
promising functional agricultural nanomaterial. The present study pro­ the SMS was removed from the cylindrical filter paper and naturally
vides a new idea for the treatment and application of SMS. dried for three days in a draft. The defatted SMS was used for further
experiments. The yield of SMS was calculated using the following
2. Materials and methods equation:
yield (%) = Wa /Wb × 100
2.1. Materials
where Wa and Wb are the weight of the SMS after and before the
The SMS used to cultivate shiitake (Lentinula edodes) was kindly defatting treatment, respectively.
provided by Social Welfare Corporation “Uizuyu” (Tottori, Japan). The
SMS was dried in the sun and preserved at room temperature until use. 2.2.1.2. Removal of lignin and hemicellulose. In this study, lignin and
Toluene and ethanol were purchased from Wako Pure Chemical In­ hemicellulose in the SMS were removed according to the method
dustries, Ltd. (Osaka, Japan). Sodium chlorite and potassium hydroxide described by Abe et al. with some modification (Abe et al., 2007). In
were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). brief, the defatted SMS was added to a flask. 1 g of SMS, 60 mL of water,
0.08 mL of acetic acid, and 0.4 g of sodium chlorite were used to remove
lignin. The flask was heated using a water bath for 1 h at 75 ◦ C, this
2.2. Isolation of CCNFC from the SMS treatment was repeated 4 times, and equal dosage of acetic acid and
sodium chlorite was added into the flask each time. Then, the solid
The detailed procedure of the CCNFC preparation is shown in Fig. 1. residue was collected by suction filtration using an aspirator and washed
with water until the filtrate turns neutral pH. The content of the SMS
2.2.1. Chemical pretreatments after delignination was gravimetrically determined.
Before the mechanical fibrillation, chemical pretreatments were
Then, the treated SMS was treated with 6 wt% of potassium hy­
performed to remove the extractives and other non-cellulose and non- droxide solution to remove hemicellulose and left at room temperature
chitin components in the SMS.
overnight. 75 g of 6 wt% potassium hydroxide aqueous solution was
used to treat 1 g of SMS. After that, the solid residue recovery and
2.2.1.1. Defatting treatment. Dried SMS was crushed using a blender (60 washing were performed using the same manner as the delignination
mesh). The SMS powder was defatted in a Soxhlet extractor with a process described above. The content of the SMS after the removal of
mixture of toluene and ethanol (2:1 (v/v)) for three days until the sol­ hemicellulose was gravimetrically determined. The treated SMS was
vent in the filter paper become colorless (Xiao et al., 2001). The heating maintained in a wet state without drying at each treatment step to avoid
of the Soxhlet extractor was used a mantle heater. After the defatting,

2
H. Li et al.
keratinization of chitin and cellulose.
2.2.2. Mechanical fibrillation
1 wt% water suspension of the chemical pretreated SMS was pre­
pared, and the suspension was passed through a grinding machine
(Super masscolloider (MKZA12-20J), Masuko Sangyo, Japan) to defi­
brillate nanofibers. The stone clearance of the grinder was set at − 1.5
mm from the zero position (the two stones slightly touched with each
other) of the two stones, and the rotational speed of the grinder was
1500 rpm. The suspension was collected after two passes of fibrillation.
Then, 1 wt% of chitin/cellulose nanofiber complex (CCNFC) water
dispersion was prepared and stored in a refrigerator at 4 ◦ C for further
use.
2.3. Characteristic evaluation of CCNFC
To verify whether CCNFC were successfully isolated from the SMS,
the characterization based on SEM, ζ-potential, FT-IR, Elemental anal­
ysis, XRD, UV–Vis, TGA, specific surface area and viscosity measure­
ments were performed.
2.3.1. Scanning electron microscopy (SEM) observation
The morphological structure of CCNFC was observed using an FE-
SEM (JSM-6700F, JEOL Ltd.). The acceleration voltage was 2.0 kV.
Before the observation, the dispersion of CCNFC was dispersed in excess
ethanol and dried in an oven at 40 ◦ C for 24 h. The dried CCNFC was
further dried in a vacuum oven at 100 ◦ C for 2 h. Then, the samples were
placed on carbon tape and then coated with a layer of platinum of about
2 nm by an automatic ion sputter coater at 10 mA for 90 s.
2.3.2. Zeta potential
To evaluate the stability of the CCNFC water dispersion, zeta po­
tential of the CCNFC water dispersion was measured using a dynamic
light scattering method (ELSZ-1000ZS, Otsuka Electronics, Osaka,
Japan) at ambient conditions (23 ◦ C). The CCNFC water dispersion was
diluted to a concentration of 0.01 wt%.
2.3.3. Fourier-transform infrared (FT-IR) spectroscopy
The FT-IR spectra of CCNFC, chitin and cellulose nanofibers were
measured using a FT-IR spectrophotometer equipped with an ATR
accessory (Spectrum 65, Perkin-Elmer Japan Ltd., Japan). The mea­
surement was performed in the wavenumber range of 550 to 4000 cm− 1
with 32 scans at a resolution of 4 cm− 1.
2.3.4. Elemental analysis
The chitin content in CCNFC was evaluated by elemental analysis
using an elemental analyzer (Elementar Vario EL III, Japan). Based on
the obtained C and N content results, the content of chitin in CCNFC was
calculated.
2.3.5. X-ray diffraction (XRD)
The crystalline structure of the CCNFC was investigated by XRD. The
diffraction profile was detected using an X-ray generator (Ultima IV,
Rigaku, Japan) at 40 kV in the goniometer scanning range of 5 to 45◦ .
The degree of crystallization was calculated from the obtained X-ray
diffraction pattern using the following equation.
I022 − Iam
Crystallinity index (%) =
I022
× 100
where I022 is the maximum intensity of diffraction of the peak at 2θ =
22◦ , and Iam is the intensity of diffraction of the peak at 2θ = 18◦ .
2.3.6. Ultraviolet-visible (UV–Vis) transmittance
The UV–vis transmittances of the CCNFC dispersion (0.1 wt%) was
measured using a UV–Vis spectrophotometer (V550, JASCO, Japan).
3
Carbohydrate Polymers 284 (2022) 119233
The wavelength range was from 200 to 800 nm.
2.3.7. Thermogravimetric analysis (TGA)
The thermal decomposition behavior of the CCNFC was investigated
using a differential thermal thermogravimetric analysis (Thermo Plus
EVO II, Rigaku, Japan). Sample was heated at 10 ◦ C/min from 25 ◦ C to
500 ◦ C under an air atmosphere.
2.3.8. Specific surface area measurement based BET method
The Brunauer-Emmett-Teller (BET) method's specific surface area of
CCNFC was measured by N2 adsorption at 77 K using an automatic
Surface Area & Pore Size Distribution Measuring System (BELSORP mini
II, MicrotracBEL Corp., Japan). A step solvent substitution using meth­
anol and tert-butyl alcohol for the specific surface area measurement of
CCNFC was performed, and lyophilized after the solvent substitution.
2.3.9. Viscosity measurement
The viscosity of the CCNFC water dispersion (1.0 wt%) was
measured using a Brookfield DV-E digital viscometer (Brookfield Engi­
neering Laboratories, Inc., Middleboro, MA, USA) at 30 ◦ C. Spindles
S61–S64 were used in the measurement. The sample volume was 100
mL. The shear rate was 0.01 to 1 s− 1. We measured the viscosity of the
CCNFC water dispersion at each shear rate for 5 min continuously. We
took the viscosity after 5 min as the final viscosity value at all shear
rates.
2.4. Biological assays of the CCNFC on plants
2.4.1. Plant materials and growth conditions
The seeds of Arabidopsis thaliana, ecotype Columbia (Col-0) were
seeded on sterilized soil (Best mix no. 3; Nippon Rockwool, Tokyo,
Japan) containing CCNFC at a concentration of 0.01, 0.1, 1, and 10 mg/
mL or without CCNFC (Control). Plants were grown under controlled
environmental conditions, with a 14 h light/10 h dark photoperiod at
25 ◦ C. Five weeks after seeding, the number of true leaves and dry
weight were measured. To evaluate plant growth, these same tests were
conducted more than 8 times with at least 6 different plants in each
treatment.
2.4.2. Reactive oxygen species (ROS) assay
Reactive oxygen species (ROS) generation production was quanti­
tatively assayed as reported by Smith and Heese (2014). One-day before
the ROS assay, leaf discs (4 mm in diameter) were obtained from 6-
week-old A. thaliana plants. Leaf discs were placed in distilled water
(DW) and kept overnight at 25 ◦ C with continuous light to reduce the
wounding effect. Then DW was discarded, and leaf discs were treated
with a suspension of CCNFC at a final concentration of 0.01, 0.1, and 1
mg/mL or without CCNFC (Control) containing 100 μM L-012 (FUJI­
FILM Wako Pure Chemical, Osaka, Japan) and 8 unit peroxidase (POD:
horseradish roots, Oriental Yeast, Tokyo, Japan). ROS production was
determined by counting photons derived from L-012–mediated chem­
iluminescence using a 2030 Multilabel Plate Reader ARVO X3 (Perki­
nElmer, MA, USA). To evaluate ROS production, these same tests were
conducted 8 times with 6 replicates in each treatment.
2.4.3. Plant pathogen infection test
Arabidopsis plants were grown on soil containing CCNFC at concen­
tration of 1 mg/mL, 0.1 mg/mL or 0.01 mg/mL or without CCNFC
(Control) for 5 weeks as aforementioned. The spores of fungal pathogen
Alternaria brassicicola isolate O-264 were prepared as described previ­
ously (Egusa et al., 2015). Droplets (10 μL) of O-264 spore suspension (5
× 104 spores/mL) were placed on the leaf surface. Inoculated plants
were kept under high humidity conditions in a moist chamber with a 14
h photoperiod at 25 ◦ C and lesion formation was observed 4 days post
inoculation. Disease severity was assessed by measuring the lesion
diameter (LD). The inoculation test was conducted 3 times with more
H. Li et al. Carbohydrate Polymers 284 (2022) 119233

than 3 different plants in each treatment. CCNFC water suspensions was shown in Fig. 2c. Despite the concen­
tration of 1.0 wt%, CCNFC exhibited a high viscosity, which means
CCNFC was well fibrillated. This is caused by the entanglement of fine
2.5. Statistical analysis
long-fiber nanofibers in water. In addition, CCNFC exhibited charac­
teristics behavior; that is, the viscosity of CCNFC decreased with rotation
The differences in the growth of plants grown on different concen­
speed. For example, when the shear rate was increased from 0.01 s− 1 to
tration of CCNFC and control were evaluated using Tukey's test. Com­
1 s− 1, the viscosity of CCNFC was reduced to approximately 1/20. This is
parison of disease severity among plants treated with different
caused by structural destruction, and the unbalance of recovery when
concentrations of CCNFC and control was performed using Chi-square
the deformation and the shear rate are applied to liquid such as sus­
test and residual analysis. Statistical data analyses were performed
pension, emulsion, and nanofiber dispersion. As the rotation speed in­
with R (version 3.6.1).
creases, the entanglement of nanofibers is broken (Li et al., 2021). The
structural change of the CCNFC water dispersion causes the nanofibers
3. Results and discussion
to separate, resulting in a decrease in viscosity. Since cellulose nano­
fibers exhibit thixotropy, they are used as ink thickeners for ballpoint
3.1. Preparation and characterization of the CCNFC
pens and thickeners for cosmetics. In addition, Yang reported that chitin
nanofibers also have thixotropy like cellulose nanofibers (Yang et al.,
We prepared CCNFC from the SMS using chemical pretreatments and
2021).
mechanical fibrillation. The SMS mainly consists of sawdust, bran, and
To confirm whether the nanofibers were successfully obtained from
some nutrients. Specifically, the SMS contains lignin, cellulose, hemi­
the SMS, the morphology of the CCNFC was investigated by SEM
cellulose from the wood, starch, protein, and other components from the
observation. Fig. 3 shows the SEM images of the raw SMS and the CCNFC
bran and nutrients. In addition, it also contains fat, chitin from mycelia,
at different magnifications. From the raw SMS SEM images, we can see
glucan, and some other polysaccharide components (Konno et al.,
rough surfaces and long tangled bundles of fibers at low magnification
2016). Chemical pretreatment is an essential step in the preparation of
(Fig. 3a) and flat surface at high magnification (Fig. 3b). On the other
cellulose nanofiber before mechanical fibrillation. The yield of the SMS
hand, many fine nanofibers with a diameter of about 10 nm were
obtained from removal of lipids, lignin, and hemicellulose were 93.4,
observed in a wide field of view from CCNFC at high magnification
45.2, and 25.2%, respectively. That is, the final extraction yield after
(Fig. 3d). This morphology was similar to cellulose and chitin nanofibers
chemical pretreatments was 25.2%. According to the previous report,
obtained by mechanical grinding (Ifuku et al., 2009; Rambabu et al.,
the cellulose content of the SMS (shiitake) is 25.4% (Konno et al., 2016),
2016). Therefore, this result, and the excellent dispersibility of CCNFC in
which is similar to our final yield after chemical pretreatments. Our
water, suggested that the nanofibers were successfully isolated from the
findings, combined with this previous study, suggested that we removed
SMS.
the non-fibrous components of the SMS via chemical pretreatments.
FT-IR spectra of chitin nanofibers, cellulose nanofibers, and the
The stability of CCNFC was evaluated by directly observing the
CCNFC were measured to verify that the CCNFC contains cellulose and
appearance of the CCNFC water dispersion (Fig. 2a), which had been
chitin nanofibers (Fig. 4a). It was found that the spectrum of the CCNFC
homogeneously dispersed for more than one month. The dispersion was
was in good agreement with the peaks derived from the chemical
milky white and viscous, similar to that of cellulose nanofibers and
structure of cellulose. In addition, characteristic shoulder peaks from the
chitin nanofibers (Ifuku et al., 2009; Rambabu et al., 2016). It can be
chemical structure of chitin, that is, OH stretching vibrations near 3482
seen that the dispersion of CCNFC was evenly dispersed in water without
and 3270 cm− 1, peaks derived from Amide I near 1661 and 1622 cm− 1,
any precipitation. This result suggests that the SMS was well converted
and amide II near 1559 cm− 1 were also confirmed. These results sug­
to nanofibers by chemical pretreatments and mechanical fibrillation,
gested that CCNFC is a complex of chitin nanofibers and cellulose
and a favorable fiber network was formed in water. In addition, to
nanofibers. Thus, it was proved that CCNFC was successfully isolated
evaluate the dispersion stability of the CCNFC in water, zeta potential
from the SMS. Moreover, elemental analysis was performed to verify the
was measured. Zeta potential is an essential indicator of the stability of a
chitin content in CCNFC. Based on the ratio of C and N, the content of
dispersion system. Generally, the absolute zeta potential higher than 20
chitin in the CCNFC was 2.6%. From the results, it was found that most
mV is stable (Sun et al., 2014). In this study, the zeta potential of the
of CCNFC is composed of cellulose derived from wood sawdust. It is in
CCNFC water dispersion was − 35.26 ± 2.70 mV at 0.01 wt%, which
agreement with the results of FT-IR spectrum and XRD profile described
indicates the CCNFC is stable in water. The UV–vis transmittance of the
later.
CCNFC was measured to evaluate the stability of the CCNFC (Fig. 2b).
Fig. 4b shows the XRD profile of chitin, cellulose, and CCNFC. XRD
The transmittance was measured at 0.1 wt%, and the transmittance of
results show of the CCNFC show two peaks derived from the crystal
CCNF at 600 nm of wavelength accounting for 9%. The viscosity of the

25 14000
a b 12000 c
20
Transmittance ( %)

Viscosity (mPa·s)

10000
15 8000

10 6000
4000
5
2000
0 0
300 500 700 900 0 0.2 0.4 0.6 0.8 1
Wave length (nm) Shear rate (s-1)

Fig. 2. Water dispersion of the chitin/cellulose nanofiber complex (a), the UV–Vis spectrum at 0.1 wt% (b), and the viscosity at 1.0 wt% (c).

4
H. Li et al. Carbohydrate Polymers 284 (2022) 119233

Fig. 3. FE-SEM images of the spent mushroom substrate (a: ×100, b: ×30,000) and the chitin/cellulose nanofiber complex (c: ×100, b: 30,000).

a b
Chitin Chitin
Absorbance

Intensity

CCNFC
CCNFC

Cellulose Cellulose
3500 2500 1500 500
5 10 15 20 25 30 35 40
Wave number (cm-1)
2 T (deg)

Fig. 4. FT-IR spectra (a) and XRD profiles (b) of chitin, chitin/cellulose nanofiber complex (CCNFC), and cellulose.

structure of natural cellulose I, i.e., peaks at around 2θ of 14.8◦ and
16.6◦ , respectively. In addition, small shoulder peaks characteristic of
the crystal structure of natural chitin, peaks derived from (020) and
(110) planes, were confirmed at around 2θ values of 9.4◦ and 19.4◦ ,
respectively. That is, CCNFC is composed of crystalline cellulose and
chitin. Therefore, it was proved from the crystal structure that CCNFC
could be isolated from the SMS. In addition, the relative crystallinity of
CCNFC was 60%. The relative crystallinity of nanofibers produced from
commercially available chitin and cellulose was 89% and 77%, respec­
tively. So, the crystallinity of CCNFC was 29% and 17% lower than
chitin and cellulose. CCNFC is characterized in that lower crystallinity
due to partial decomposition by fungi compared with common wood-
derived cellulose nanofibers. In addition, as mentioned above, CCNFC
contains glucan, so it is believed that glucan may reduce the relative
crystallinity of CCNFC.
The thermal stability of CCNFC was examined by TGA (Fig. 5). Due
to water evaporation, there was no significant difference in the first
stage decomposition of CCNFC at 30 to 100 ◦ C. CCNFC begins to degrade
at about 250 ◦ C and reaches the maximum decomposition in the second
stage around 340 ◦ C. As can be seen, CCNFC is stable up to around

5
H. Li et al.
0
-20
Weight loss (%)
-40
-60
-80
-100
0 100 200
Temperature ( )
Fig. 5. Thermogravimetric analysis
fiber complex.
250 ◦ C. This thermogram is in good agreement with that of cellulose and
chitin nanofibers (Larbi et al., 2018; Rambabu et al., 2016).
Specific surface area of CCNFC was measured by N2 adsorption
(Fig. 6). The type of nitrogen adsorption isotherm of CCNFC is type II,
indicating that CCNFC is a typical non-porous or microporous structure,
consistent with the results of microscope observation. In other words,
CCNFC is a nanofiber structure. The specific surface area of CCNFC was
198.2 m2/g. The large specific surface area suggests that purified SMS
has been converted to nanofibers effectively.
As can be seen from the above characterization results, CCNFC shows
good dispersion, high viscosity, high thermal stability, and large specific
surface area. In a previous study, Konno et al. isolated cellulose nano­
fibers from SMS and prepared cellulose nanofiber film; thus, our find­
ings, combined with theirs study, suggested that SMS may be a
promising candidate source of nanofiber. In addition, we have reported
that chitin nanofibers could improve the nitrogen absorption efficiency
of tomatoes, promote the growth of tomatoes, and induce disease
resistance of plants (Egusa et al., 2019, 2020; Parada et al., 2018).
Because the CCNFC contains chitin nanofibers, we predicted that CCNFC
might have related biological activities similar to chitin nanofibers on
plants. Specifically, we evaluated the biological activities of CCNFC on
plants, including disease resistance induction and growth promotion
properties. The detailed results of the biological assays are described in
the following section.
600
Absorbed volume (cm3/g)
˖ GHVRUSWLRQ
˖ DGVRUSWLRQ
400
200
0
0 0.5
Relative pressure (p/p0)
Fig. 6. The adsorption and desorption profile of chitin/cellulose nano­
fiber complex.
Carbohydrate Polymers 284 (2022) 119233
3.2. Effects of nanofibers on plant growth and disease resistance
A. thaliana plants were grown on CCNFC contained soil and the
impact of CCNFC on plant growth was evaluated. Supplementing CCNFC
at 0.1 and 0.01 mg/mL moderately increased the number of leaves
(Fig. 7a) and shoot dry weight (Fig. 7b) compared with CCNFC un­
treated control plant. On the other hand, plant growth was decreased by
treatment with CCNFC at concentration of 10 and 1 mg/mL. Especially,
the application of CCNFC at high concentration (10 mg/mL) severely
inhibited the plant growth. These results indicated CCNFC was effective
in increasing plant growth which requires using optimal concentration.
In addition to growth, we examine the effect of CCNFC on disease
resistance in plants. ROS production is one of the first disease resistance
responses in plants (Qi et al., 2017). Treatment with CCNFC induced
ROS production in Arabidopsis (Fig. 8a). CCNFC treatment at a concen­
300 400 500
tration of 1 mg/mL showed higher production of ROS than the others.
ROS production was slightly induced by 0.1 mg/mL CCNFC treatment
compared with control treatment, and not observed by treatment of
curve of chitin/cellulose nano­ 0.01 mg/mL CCNFC. These results indicated that CCNFC induced ROS
production in a dose-dependent manner.
To evaluate the effect of CCNFC on induced disease resistance
further, Arabidopsis plants grown on CCNFC-containing soil were inoc­
ulated with fungal pathogen A. brassicicola. As shown in Fig. 8b, treat­
ment of 1 mg/mL CCNFC significantly reduced disease severity (3 < LD
< 5 mm and 5 mm < LD) and symptom became mild. Similarly, treat­
ment with 0.1 mg/mL of CCNFC showed lower disease severity (3 < LD
< 5 mm) than control treatment. On the other hand, treatment of 0.01
mg/mL CCNFC had no protective effect. These results indicated that
CCNFC can induce systemic resistance when mixed with soil.
Because CCNFC mainly consists of cellulose and chitin nanofiber,
chitin would play a role in the enhancement of plant growth and disease
resistance. The beneficial effects of chitin on plant defense and growth
have been well documented (Shahrajabian et al., 2021; Sharp, 2013).
We previously reported that nanofiber prepared from chitin can increase
plant growth, ROS production, and resistance against pathogen infection
in not only A. thaliana but also various crops such as rice, cabbage,
strawberry, and tomato (Egusa et al., 2015, 2019, 2020; Parada et al.,
2018). On the other hand, the knowledge about the role of cellulose and
its derivatives in plant growth and disease resistance is so limited: it has
only been reported that the cellulose degradation product, cellobiose,
induces defense-like responses and increases plant growth in A. thaliana
(de Azevedo Souza et al., 2017). Thus, the involvement of cellulose, a
major component of CCNFC, in plant growth and disease resistance is
still controversial.
Typically the treatment of a high concentration of plant defense
inducer negatively affected plant growth (Gómez-Gómez et al., 1999;
Parada et al., 2018). Likewise, the effects of CCNFC on plant growth
were positive and negative at low (0.1 and 0.01 mg/mL) and high (1 and
10 mg/mL) concentrations, respectively. These results suggested that a
tradeoff between defense and growth in plants was occurred by CCNFC,
which is a generally known phenomenon to make a balance to optimize
plant fitness (Huot et al., 2014). The molecular mechanism underlying
the tradeoff caused by CCNFC is hitherto not clear. As a result, the effects
of CCNFC are expected to apply to other crops, which infers CCNFC
would be a promising nanomaterial in agriculture. For practical use of
CCNFC in agriculture, further study is needed to elucidate the mecha­
nism how CCNFC simultaneously induces both growth promotion and
disease resistance in plants.
4. Conclusion
1 This study successfully prepared a novel nanomaterial, chitin/cel­
lulose nanofiber complex (CCNFC), from the SMS. Its biological activity
assays of the CCNFC were also performed to investigate the usefulness of
CCNFC as a functional agricultural nanomaterial. Our results indicated
the potential of CCNFC as a functional nanomaterial applied in
6
H. Li et al. Carbohydrate Polymers 284 (2022) 119233

Fig. 7. The effect of CCNFC on the growth of Arabidopsis thaliana plants. Five weeks after seeding, the number of leaves (a) and shoot dry weight (b) were measured.
Different letters indicate statistically significant differences (Tukey's test, P < 0.05).

Fig. 8. The effect of CCNFC on disease resistance in Arabidopsis thaliana. (a) Generation of reactive oxygen species (ROS) by CCNFC treatment. The values represent
the average and standard errors of 8 independent experiments with 6 replicates in each treatment. (b) CCNFC treatment induces disease resistance to Alternaria
brassicicola. Arrowheads indicate statistically significant different frequency distributions (high or low) of the disease severity among treatment (Chi-square test and
residual analysis, P < 0.05).

agriculture effectively. Moreover, we expect that the CCNFC may have Conceptualization, Methodology, Funding acquisition, Writing – orig­
other biological activities. Our findings provide a new way to treat and inal draft. Shinsuke Ifuku: Conceptualization, Methodology, Writing –
utilize the SMS. review & editing, Funding acquisition.

Ethical approval
Declaration of competing interest

Not applicable.
All authors declare that they have no conflicts of interest.

Ethical standards
Not applicable.
Human and animal rights
This is not research involving human participants and animals.
CRediT authorship contribution statement
Hujun Li: Writing – original draft, Investigation. Saori Yoshida:
Investigation. Naofumi Mitani: Investigation. Mayumi Egusa: Inves­
tigation, Writing – original draft. Momoko Takagi: Investigation,
Writing – original draft. Hironori Izawa: Writing – review & editing.
Teruyuki Matsumoto: Resources, Supervision. Hironori Kaminaka:
Acknowledgements
We would like to thank Dr. Hiroshi Otani (Tottori University) and
Social Welfare Corporation “Uizuyu” for providing the plant-pathogenic
fungus and the shiitake SMS, respectively.
Formatting of funding sources
This research was supported by grants from the project of the NARO
Bio-oriented Technology Research Advancement Institution (Advanced
integration research for agriculture and interdisciplinary fields), and a
Grant-in-Aid for Scientific Research (B) from the Japan Society for the
Promotion of Science (JSPS) (JSPS KAKENHI Grant Number
16H02998).

7
H. Li et al. Carbohydrate Polymers 284 (2022) 119233

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