pubs.acs.

org/acsagscitech Article

Iron-Carbon Nanofibers Coated with Acylated Homoserine Lactone

Enhance Plant Growth and Suppress Fusarium Wilt Disease in Cicer
arietinum by Modulating Soil Microbiome
Komal Pandey, Nishith Verma,* and Govind Gupta*
Cite This: ACS Agric. Sci. Technol. 2022, 2, 311−322 Read Online

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ABSTRACT: The agricultural soil microbiome plays a key role in crop productivity, nutritional quality, and suppressing soil-borne
plant pathogens. In the current study, a nanocomposite biofertilizer based on acylated homoserine-coated iron-carbon nanofibers is
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developed that potentially improves the soil rhizomicrobiome. The 16S RNA-based metagenome sequencing of soil bacteria shows
that the abundance of specific bacterial genera (e.g., Lactobacillus, Bacillus, and Pseudomonas) is potentially improved in the
nanocomposite (1 g/kg)-treated plant−soil system after 30 days. As a result, the prepared nanocomposite promotes growth,
nutritional quality, and suppresses the fungal infection (Fusarium oxysporum f. sp. cicero) in chickpea plants. In addition, the data
show an increased total nitrogen content in nanocomposite-amended soil collected after 30 days of plant growth, which is the
indicator of an increase in soil nitrogen fixation potential. Commonly, nitrogen content in soil is maintained by rhizobacteria;
therefore, the increased soil nitrogen content is linked to the improved soil microbiome. In the plant growth experiment, a
statistically significant (p < 0.05) increase is observed in the wet biomass, root length, shoot length, and chlorophyll-protein contents
of the plants. The developed nanocomposite biofertilizer in this study has novel implications in sustainable agriculture production.
KEYWORDS: AHL/Fe-CNF NC, nanobiofertilizer, sustainable agriculture, rhizomicrobiome, pathogen defense

■ INTRODUCTION
The agriculture sector is facing numerous challenges globally
because of climate variation, which in turn leads to a number of
adverse radical events in terrestrial soil, for example, drought,
nutrient limitations, and altered rhizospheric microbial
community (rhizomicrobiome), including microbial func-
tions.1,2 Rhizomicrobiome has critical functions in farming
and agricultural production. Thus, any adverse effects can
directly affect the food resources and their nutrient content.3−6
Consumption of food products grown in the soil excessively
treated with chemical fertilizers may impose the risk of mortal
diseases, especially cancer.7 On the other hand, the increasing
global population demands a sustained increase in crop
production in order to achieve a hunger-free community. For
sustainable agriculture applications, it is essential to develop
multipronged technologies that can counteract the impact of
climatic changes, and at the same time, improve agricultural
production.
Nanotechnology has a vast scope in agricultural sectors, for
example, nanocarriers for pesticides and fertilizers, and nano-
sensors for plant pathogens.8,9 Nanomaterial-based fertilizers
and pesticides can be promising next-generation fertilizers that
can improve crop production in an environmentally safe
way.10−13 Some metal nanoparticles have been shown to trigger
positive effects as micronutrients on crop production.14−17
Dimkpa et al. studied the effect of oxides of zinc, copper, and
boron on the growth of soybean plants and compared their effect
with that of their bulk counterparts.18 The study showed that the
mixed oxide nanoparticles, but not their bulk counterparts,
stimulated shoot growth, flower formation, shoot biomass, and
accumulation of nitrogen and potassium in shoots. In addition,
Karny et al. have shown an effective way of treating metal
nutrient deficiency in plants by introducing lipid nanoparticles
loaded with iron (Fe) and magnesium.19 In contrast, some
studies have shown that exceeding a threshold concentration of
metallic nanoparticles can trigger toxic effects in plants or the
surrounding ecosystem.20,21 In line with this finding, Mortimer
et al. performed a transcriptomics-based study that indicated
certain engineered nanomaterials (e.g., cerium oxide, carbon
nanotubes, and carbon black) interfering with plant-bacterial
signaling, as evidenced by the suppressed upregulation of genes
induced by root exudates and downregulation of genes encoding
transport RNA that facilitates nodulation signaling.22
Recent studies have indicated that the delivery of microbial
consortia mediated by nanomaterials and the enhancement of
soil microbial communities can retain most of the natural
habitats, and at the same time, enhance crop production.23,24
Timmusk et al. have shown that coexposure of titanium dioxide
nanoparticles and plant growth-promoting rhizobacteria
(PGPR) to winter wheat (Triticum aestivum cv. Stava) enhances
the seedling biomass and resistance to plant pathogen and
abiotic stresses.25 The aforesaid study has demonstrated that
Received: August 30, 2021
Revised: February 21, 2022
Accepted: March 7, 2022
Published: March 17, 2022

© 2022 American Chemical Society https://doi.org/10.1021/acsagscitech.1c00217

311 ACS Agric. Sci. Technol. 2022, 2, 311−322
ACS Agricultural Science & Technology
Table 1. Different Groups Used for Seed Germination and Plant Growth Experiments
titanium dioxide nanoparticles enhance the colonization of
PGPR bacteria in plant roots, which in turn supports plant
growth. Liu et al. introduced nanoscale zero-valent iron (nZVI)
in pentachlorophenol-contaminated paddy soil and demon-
strated that nZVI decreased the pentachlorophenol content and
increased rice production synergistically.24 The authors also
showed that the degradation of pentachlorophenol in nZVI-
amended soil was facilitated by the rhizospheric microbial
community.
Healthy soil is a key for sustaining the health of plants and the
environment.5 The microbial community (also known as the
microbiome) is one of the dominant factors in soil health, in
addition to the soil organic matter, soluble and reduced
nitrogen, soil topography, water holding capacity, and climate.26
Therefore, any perturbation in the microbiome population can
lead to serious health effects on plants and the environment.3
Short-chain N-acyl homoserine lactone (AHL) is a quorum-
sensing signal molecule secreted in soil by Gram-negative
bacteria.27 AHL is closely linked to building up the healthy soil
rhizomicrobiome (microbiome surrounding plant roots) that
further regulates the plant development and suppression of soil-
borne pathogens.28,29 In a previous study, we used carbon
nanofibers (CNFs) as a carrier agent for plant micronutrient Fe
and synthetic AHL.30 We experimentally demonstrated that the
nanocomposite of AHL and Fe-CNFs (denoted as AHL/Fe-
CNFs) enhanced plant growth and resistance to fungal
pathogens, but the mechanism was unknown. Therefore, the
current study was performed to explore the possible mechanism
of growth induction in chickpea plants grown in the soil treated
with the AHL/Fe-CNF nanocomposite. Considering that AHL
is a bacterial communication signal that regulates plant growth
by establishing plant−microbe interactions in the rhizo-
sphere,31,32 we hypothesize that the nanocomposite treatment
in soil may influence the soil rhizomicrobiome, which in turn
triggers plant growth and defense to pathogens. We applied a
metagenome-based approach to study rhizomicrobiome of the
soil collected after 30 days of plant growth with/without the
application of the AHL/Fe-CNF nanocomposite and evaluated
312
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its influence on rhizomicrobiome, nitrogen assimilation, and the
growth of plants.
■
MATERIALS AND METHODS
Biological and Chemical Materials. Iron nitrate (Fe-
(NO3)3.9H2O, >95%), hydrochloric acid (HCl, 36%), sulfuric acid
(H2SO4, 98%), boric acid (H3BO3, 99%), potassium permanganate
(KMnO4, 98%), sodium acetate (C2H3NaO2, 99%), dipotassium
hydrogen phosphate (K 2 HPO 4 , 99%), triammonium citrate
(C6H17N3O7, 97%), magnesium sulfate heptahydrate (MgSO4.7H2O
98%), manganese sulfate tetrahydrate (MnSO4.4H2O, 98%), poly-
sorbate 80 (Tween 80), bromocresol green (C12H14Br4O5S), and
methyl red (C15H15N3O2) were purchased from Merck (Germany). N-
Butanoyl-L-homoserine lactone (C4-HSL) was purchased from Sigma-
Aldrich (Germany). Sodium hydroxide (NaOH) was purchased from
Fisher Scientific (India). Bovine serum albumin, potato dextrose agar,
protease peptone, HM peptone B (beef extract), yeast extract, dextrose,
Gram’s staining kit, and agar powder were purchased from HiMedia
Pvt. Ltd. (India). A culture of the fungus Fusarium oxysporum f. sp. ciceri
was kindly donated by the ICAR-Indian Institute of Pulses Research
(Kanpur, India). Hydrogen (H2), nitrogen (N2), and acetylene (C2H2)
gases were purchased from Sigma Gases (India). Cicer arietinum seeds
(variety: IPF 4-9) were a kind gift from the Horticulture facility at IIT
Kanpur (Kanpur, India). All reagents were of high purity grade (>98%)
and used without further purification. All solutions were prepared in
ultrapure Milli-Q water (Elix Milli Q system, USA).
Synthesis of Iron-Carbon Nanofibers. Fe-CNFs were synthe-
sized by following the previously described protocol, with slight
modifications.23,33 Briefly, Fe(NO3)3.9H2O salt was subjected to
calcination at 400 °C for 4 h, using nitrogen flow of 200 standard
cubic centimeter per min (sccm) to convert metal nitrate to metal
oxides, i.e., Fe(NO3) to FeO in a vertical furnace (Mahendra scientific
inst. Mfg. Co., Kanpur, India). The reduction of FeO to zero-valent Fe
was carried out at 660 °C for 2 h, using hydrogen flow at 200 sccm.
Chemical vapor deposition (CVD) was performed under the flow of
C2H2 (35 sccm at 660 °C for 1 h) to grow CNFs using the zero-valent
Fe nanoparticles as the catalyst for the CVD reaction.
Interaction of Fe-CNFs and AHL. Approximately 0.5 g of Fe-
CNFs was soaked in 50 mL of Milli-Q water. Next, 0.1 μM AHL was
added to the Fe-CNF suspension and the suspension was allowed to
interact for 1 h at room temperature (∼25 °C) under constant stirring.
The AHL/Fe-CNF nanocomposites (referred as NCs hereafter) were
https://doi.org/10.1021/acsagscitech.1c00217
ACS Agric. Sci. Technol. 2022, 2, 311−322
ACS Agricultural Science & Technology
separated by centrifugation (7500 rpm for 0.5 h) and redispersed in
water before adding it to the soil for plant growth experiments.
Hydrodynamic Size and Zeta Potential Measurements. The
suspensions of Fe-CNFs and NCs were prepared in Milli-Q water, and
the hydrodynamic size and zeta potential were determined using a
particle size analyzer (Microtrac Bluewave, USA) and a Zetasizer Nano
ZS90 (Zetasizer, Malvern, UK), respectively.
Scanning Electron Microscopy and Energy-Dispersive X-ray
Spectroscopy Analysis. The shape and elemental composition of the
synthesized nanocomposite fertilizers were determined by using
scanning electron microscopy (SEM) attached with energy-dispersive
X-ray spectroscopy (EDS) (JSM-7100F, JEOL, USA). The samples for
SEM-EDS analysis were prepared by directly coating them on stubs,
followed by sputter coating (20 nm) with gold (Au). The plant samples
for SEM-EDS analysis were prepared as described earlier.30 Briefly,
samples were prefixed with 4% glutaraldehyde, followed by a series of
dehydration with gradients (10−100%) of ethanol. Dehydrated
samples were then loaded in SEM stubs using carbon tape and
subjected to conductive sputter coating (20 nm) with gold. The
samples were analyzed under field-emission SEM (MIRA-3 TESCAN,
Brno, Czech Republic).
Fourier Transform Infrared Spectroscopy. Functional groups in
Fe-CNFs with/without AHL coating were determined using Fourier
transform infrared (FTIR) spectroscopy. The samples for FTIR
analysis were prepared by dispersing Fe-CNFs, AHL, and NCs in
Milli-Q water. The samples were scanned at the wavenumbers 4000−
400 cm−1 using the attenuated total reflectance FTIR (Bruker, Tensor
27). The water was run as a background to avoid interference from
water molecules in the spectra. The spectra were plotted over the range
of 4000−400 cm−1 wavenumbers using OriginPro software.
Experimental Plan. As shown in Table 1, the study was performed
in two experimental setups (seed germination and plant growth) with
four individual treatments in each setup.
Seed Germination Experiments. All seed germination and
radicle growth experiments were carried out in the agar medium.
Cicer arietinum (chickpea) seeds were soaked overnight in water and
sterilized for 10 min, using 70% (v/v) ethanol on the following day, and
then ethanol was evaporated in an oven for 1 h. The agar medium was
prepared by dissolving 15 g/L of agar powder in Milli-Q water, followed
by steam-sterilization in an autoclave. The various treatments (NC, Fe-
CNF, and AHL), and control (water only) were added to the agar plates
before they were solidified. Thereafter, 10 seeds were transferred to
each Petri dish. The seeds were allowed to germinate in the Petri dishes
under a BOD incubator (Mahendra Scientific inst. Mfg. Co., Kanpur,
India) at 25 °C. The number of germinated seeds in control and
treatment groups was recorded every day for six consecutive days. On
the sixth day, the radicle and hypocotyl growth of the seeds was also
measured and recorded. Photographs of the germinated seeds were
captured using a digital camera on day 6. The data were plotted in the
form of the germination index (GI) and radicle growth. GI was
calculated by applying the formula suggested by Ashfaq et al.:16
(number of germinated seeds)
GI = + ...
(first day of count)
+ (number of germinated seeds)/(final day of count)
Plant Growth and Fungal Stress Experiments. Sandy-clay soil
was collected from the Indian Institute of Technology Kanpur (Kanpur,
India). The soil was sieved using a 2 mm (sieve opening size) mesh. The
sieved-separated soil was kept at 30 °C for 24 h to acclimatize the soil
sample to normal climatic temperature in the geographical region.
Thermogravimetric analysis (TGA, Mettler Toledo) showed a
negligible (0.4%) weight loss from such soil sample over 25−30 °C
(Supporting Information (SI) Figure S1). The soil was characterized
for the texture and elemental contents using a hydrometer (Apollo,
India) and X-ray fluorescence spectroscopy (WD-XRF, Rigaku, Japan),
respectively. The results of texture analysis and elemental composition
are provided in the Supporting Information Figures S2 and S3,
respectively. The texture analysis of the soil showed a dominant
presence of silt (85%) and sand (14%), including clay (1%) (Figure
313
pubs.acs.org/acsagscitech Article
S2). The XRF analysis for elemental compositions showed a dominant
presence of Si, Al, and Fe in major elements (Figure S3). Planter pots of
1 kg capacity were half-filled (500 g) with dried soil (bulk density of soil
1.30 g/cm3). Each pot was labeled appropriately and supplied with the
following treatments after dispensing them in 50 mL of Milli-Q water:
(1) Milli-Q water alone (control), (2) Fe-CNF (1 g/kg), (3) AHL (0.1
μM), and (4) NC (1 g/kg). Each treatment and control were prepared
in triplicate (n = 3). On the following day, 10 sterilized seeds were sown
and supplied with 50 mL of Milli-Q water every alternate day for 30
days to keep the moisture content approximately constant throughout
the experiment. The plants were grown and maintained under standard
conditions during the natural dark−light cycle (14 h dark−10 h light).
No further treatment of NCs was given throughout the experiment.
Next, plants were harvested after 30 days, and growth measurements
were performed by analyzing the parameters: shoot length, root length,
wet biomass, protein content, and chlorophyll contents. Atomic
absorption spectroscopy (AAS) analysis and SEM-EDS were
performed to measure the uptake and translocation of Fe-CNFs in
the plants. In addition, total nitrogen (N) content in the soil was
estimated after harvesting the plants from control and each treatment.
For microbiome study, 1 g of the soil sample was collected from each
replicate (n = 3) for control and treatment groups, and then the samples
were pooled and stored at −40 °C in a deep freezer until use.
In a separate set of experiments, but with the same control and
treatments as above, the soil was infected (at day 0 of the experiment)
with a fungus Fusarium oxysporum ciceri. Fusarium oxysporum ciceri was
cultured in potato dextrose agar medium for 3 days in an incubator at 30
°C. Next, the fungal mycelium was collected and dispersed in 50 mL of
Milli-Q water by vortexing. Plants were grown up to 30 days with fungus
alone (control) and in the combination of fungus and the respective
treatment: AHL (0.1 μM or 1.6 mg/kg-soil), Fe-CNF (1 g/kg-soil), and
AHL/Fe-CNF NC (1 g/kg-soil). Plant growth was measured in the
same way as described above for nonfungal-treated plants. In addition,
photomicrographs were captured using a digital camera to visualize any
phenotypic anomalies because of fungus infection.
Protein Analysis. Approximately 0.5 g of ground plant shoot from
each sample (control, Fe-CNF, AHL, and NC) was incubated in 5 mL
of phosphate buffer solution for 60 min at ∼25 °C. Following
incubation, samples were centrifuged at 1250 g for 15 min and the
supernatant was collected. Next, 1 mL of supernatant was mixed with 5
mL of alkaline copper solution and 500 μL of Folin’s reagent followed
by incubation for 30 min and in the dark. The absorbance of protein in
the solution was determined at 660 nm wavelength, using a UV−vis
spectrophotometer (Varian Cary 100, Germany). Bovine serum
albumin was used for calibration of the standard curve with correlation
R2 = 0.982.
Chlorophyll Analysis. Approximately 0.5 g of ground plant leaves
from each sample (control, Fe-CNF, AHL, and NC) were incubated in
10 mL of acetone (80%) at 4 °C for 12 h. After incubation, the samples
were centrifuged at 1250 × g for 15 min. Approximately 4 mL of
supernatant was collected and the absorbance of chlorophyll was
determined at the wavelengths 645 and 663 nm using a UV−vis
spectrophotometer (Varian Cary 100, Germany). The following
formulas were applied to determine chlorophyll a and b contents in
plant leaves:16
chlorophyll a (mg/g)
= (12.7 × A 663) − (2.6 × A 645) × acetone (mL)
/leaf sample (mg)
chlorophyll b (mg/g)
= (22.9 × A 645) − (4.68 × A 663) × acetone (mL)
/leaf sample (mg)
total chlorophyll (mg/g) = chlorophyll a + chlorophyll b
Optical Microscopy. The transverse sections of the root, shoot,
and leaves were prepared after 30 days of treatment. The respective
https://doi.org/10.1021/acsagscitech.1c00217
ACS Agric. Sci. Technol. 2022, 2, 311−322
ACS Agricultural Science & Technology pubs.acs.org/acsagscitech Article

sections were then placed on glass slides and examined under bright-
field microscopy at 400× magnification using Leica DM2000 (Wetzlar,
Germany). The black granules were located in the vascular bundles to
check the uptake and translocation of Fe-CNFs in plants through the
root, shoot, and leaf.
AAS Analysis for Iron Content. Approximately 0.5 g of the root,
shoot, and leaves were dried in a hot air oven for 24 h at 80 °C. On the
following day, samples were acid-digested using aqua regia (1:3 ratios of
HNO3:HCl) for 72 h at 85−90 °C in a fume hood. After complete
digestion, samples were diluted using 2% HNO3. Fe content of the
samples was analyzed using AAS (AA-420 Varian, USA).
Total Nitrogen Content Estimation in Soil. Total nitrogen
content (sum of organic and inorganic (ammonia and ammonium)
contents) of soil was determined after 30 days of plant growth in the
presence and absence of NCs. Approximately 5 g of soil was collected
from each pot in control and treatment groups after 30 days of plant
growth. Next, the soil sample was placed in a digestion tube and wetted
by adding ultrapure MQ water. The tube was then placed in an
instrument and 25 mL of 0.32% KMnO4 and 25 mL of 4% NaOH were
added one after another. Next, tunes were loaded in a distillation system
using a slider. Approximately 25 mL of boric acid (2.5%) together with
an indicator (bromocresol green and methyl red) was prepared in a
separate 250 mL glass conical flask and dipped in the receiving end of
polysorbate 80 (Tween 80), 2 g of dipotassium hydrogen phosphate, 2
g of triammonium citrate, 0.1 g of magnesium sulfate heptahydrate, 0.05
g of manganese sulfate tetrahydrate, and 12 g of agar dissolved in 1 L of
Milli-Q water. The prepared medium was autoclaved and plated in
sterile Petri dishes to solidify. In parallel, 1 g of collected soil after 30
days of plant growth experiment was serially diluted up to 100,000
times. One hundred microliters of diluted soil from each dilution was
spread in the prepared MRS plates. The plates were left in an incubator
at 37 °C for 24 h. After incubation, bacterial colonies were counted
manually for the control and each treatment. The results were plotted as
a colony-forming unit (CFU) per milliliter of each sample. An
appropriate dilution factor was applied while calculating CFU/mL.
Gram staining of the bacteria in colonies was performed to check the
Bacillus property of the isolated bacteria including its shape.
Statistical Analysis. The statistical significance of the differences
among three treatments (AHL, Fe-CNF, and AHL/Fe-CNF) and the
control was determined using the one-way ANOVA and Dunnett’s or
Tukey’s post hoc tests. Dunnett’s post hoc test was applied to calculate
the statistical difference between control and individual treatments;
however, Tukey’s tests were applied to calculate statistical differences
among different treatments. GraphPad Prism 6 software (San Diego,
CA, U.S.) was used to perform statistical analysis. Differences were
considered to be statistically significant at p < 0.05.

■
the distillation system. Subsequently, distilled ammonia gas was
collected in the receiver end in a solution. Next, titration was performed
in the collected distillate using 0.2 N H2SO4 until the color of the RESULTS AND DISCUSSION
solution turned pink. Nitrogen content in each sample was calculated Synthesis and Characterization of Nanocomposites.
using a KjelTRON nitrogen estimation system (KDIGB 4 M, Fe-CNFs were synthesized as per the procedure described in the
KjelDISTA, India). Total nitrogen content (kg/hec) in soil was
previous studies.23,33 The synthesized Fe-CNFs (1 g/L) were
calculated from titration values by applying the following formula:
allowed to interact with AHL (0.1 μM) for 1 h in MQ water. The
nitrogen (kg/hec) = [14 × (normality of acid)
AHL-coated Fe-CNFs, termed AHL/Fe-CNFs or NCs were

× (actual titrate value) × 2.20

× 106 /sample weight (g) × 1000]

Soil Microbiome Screening. DNA Extraction, PCR, and 16S

rRNA Sequencing of Soil. DNA extraction and purification were
performed using an EXpure DNA isolation kit (Bogar Bio Bee Stores
Pvt. Ltd., India). The isolated DNA concentration was measured using
a Qubit 3.0 fluorometer (Thermo Fisher Scientific). Next, a polymerase
chain reaction (PCR) was performed using 5 μL of isolated DNA. The
PCR reaction was set in by adding forward-reverse primers (27F: 5’
AGAGTTTGATCMTGGCTCAG 3′ and 1492R: 5’ TACGGY-
TACCTTGTTACGACTT 3′) and Taq Master Mix. PCR was
performed using thermal cycling conditions. After the amplification
reaction, the PCR product was purified by removing unincorporated
PCR primers and dNTPs by using the Montage PCR Clean-Up kit
(Sigma-Aldrich, India). The quality, quantity (app.100 ng/μL), and
formulation of the PCR product were checked using a fluorometer.
Nanopore sequencing (accuracy 99.9%), a third-generation
sequencing was performed by using 1 μg of DNA template, according
to the laboratory’s instructions. Next, bioinformatics analysis on the
sequenced data was performed by using EPI2ME 16S analysis, which
allowed us to perform genus-level identification from single reads, with
detailed investigations at species and subspecies levels. The phylogeny
analysis of the query sequence with the closely related sequence of
BLAST results was performed, followed by multiple sequence
alignment. The workflow is designed to BLAST base called sequence
against the NCBI 16S bacterial database which contains 16S sequences
from different organisms. Each read is classified based on % coverage
and identity. The bacteria genera identified in 16S RNA sequencing
were plotted as a heatmap based on the most to least abundance in soil.
In addition, the most abundant bacterial genus was plotted for species
richness in a separate heatmap. Figure 1. Characterization of the prepared NCs. (a) Charge-based
Isolation and Screening of Lactobacillus spp. from the Soil. interactions between AHL (0.1 μM) and Fe-CNFs (10 g/L) as
Lactobacillus bacterial sp. was isolated and screened using a specific evidenced by changes in the zeta potential. (b) FTIR spectra of Fe-
selective MRS medium (de Man, Rogasa and Sharpe). MRS medium CNFs before and after interaction with AHL molecules, indicating the
was prepared by mixing 10 g of protease peptone, 10 g of HM peptone, binding of AHL with Fe-CNFs. Refer to Supporting Information figures
5 g of yeast extract, 20 g of dextrose, 5 g of sodium acetate, 1 g of for the additional characterization data.

314 https://doi.org/10.1021/acsagscitech.1c00217
ACS Agric. Sci. Technol. 2022, 2, 311−322
ACS Agricultural Science & Technology
Figure 2. Seed germination and radicle growth of chickpea seeds after 6
days of treatment with NCs. (a) Optical micrographs of germinated
seeds in the control, Fe-CNF, AHL, and NC at day 6. (b, c) GI and
radical growth of seeds at day 6, respectively. Data presented are mean
± SD (n = 3). Statistical significance was calculated by applying one-
way ANOVA and Tukey’s post hoc test. *p < 0.05 and **p < 0.01 were
considered significant with respect to the control. #p < 0.05 and ##p <
0.01, indicate significance among individual treatments.
pelleted by centrifugation and resuspended in 50 mL of MQ
water. The morphology and elemental composition of Fe-CNFs
and NCs were determined by performing SEM and EDS,
respectively. Bright carbon fibers were visible in the SEM images
of both, pre-and postfunctionalized materials (Figure S4a,b).
EDS analysis was performed to confirm the presence of Fe in the
synthesized materials. As shown in Figure S4c,d, 16 and 8% (w/
w) of Fe were measured in Fe-CNFs and NCs, respectively.
Next, the hydrodynamic size of the Fe-CNF and NC
suspensions in water was determined using a particle size
analyzer. As shown in Figure S4e,f, the particle sizes of both
materials were found to be in the range of 10−100 nm.
Electrostatic interactions play an important role in the
complex formation between the oppositely charged nano-
particles and biological ligands.34 As depicted in Figure 1a,
positively charged moieties on AHL may enable electrostatic
interactions with the negatively charged Fe-CNFs. To
investigate the interactions between Fe-CNFs and AHL, we
performed the zeta potential and surface functional group
measurements in Fe-CNFs post interaction with AHL, using a
zetasizer and FTIR spectroscopy, respectively. As shown in
Figure 1b, the negative z-potential of Fe-CNFs (−22.0 mV) was
decreased to −18.0 mV after the material interacted with AHL,
i.e., in NCs, which evidenced an electrostatic interaction
between AHL and Fe-CNFs. Furthermore, FTIR data
315
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Figure 3. Uptake or translocation of NCs in plants and nitrogen (N)
content in the soil after 30 days of treatment (1 g-NC/kg-soil). (a) Fe
content in the root, shoot, and leaves of plants indicates uptake and
translocation of NCs. (b) N content in the soil was measured at day 30
after harvesting the plants. Data presented are mean ± SD (n = 3).
Statistical significance was calculated by applying one-way ANOVA and
Tukey’s post hoc test. *p < 0.05 and ***p < 0.001 were considered
significant with respect to the control. ###p < 0.001 indicates significance
between Fe-CNF and NC treatments.
confirmed the presence of AHL on the surface of Fe-CNFs
(Figure 1c). An amide group is one of the characteristic
signatures of AHL, which was detected and distinguished at
1575 cm−1 in the spectra of AHL as well as NCs; however, it was
absent in the spectra of Fe-CNFs alone, confirming the
successful binding of AHL with Fe-CNFs.
Nanocomposite Boosts Seed Germination and Seed-
ling Growth in Chickpea. Germination of seeds and seedling
growth is the first critical step in plant growth. Ma et al. have
shown that relatively faster germination can lead to enhanced
growth and production in perennial grass (Leymus chinensis)
after seed priming with gibberellic acid-3 (GA3).35 Zvinavashe
et al. have demonstrated that the application of silk fibroin- and
trehalose-coated rhizobacteria on seed surfaces can boost
germination and seedling growth, which has a positive effect
on the crop yield.36 Therefore, we first looked at whether or not
NCs show a positive effect on seed germination and seedling
growth. As shown in Figure 2, an improvement was recorded in
seed germination and seedling growth after 6 days of treatment
with NCs, as compared to that with control (seeds grown
without NCs) or the AHL or Fe-CNF treatment alone. A visual
difference can be seen between the seedling growth in
photomicrographs taken after 6 days of seed germination
with/without NCs (Figure 2a). Furthermore, the GI of chickpea
(Cicer arietinum) seeds was calculated, which was significantly (p
< 0.05) higher in the seeds grown with NCs (2.1 ± 0.2) than
control (0.9 ± 0.37) or individual treatments (Fe-CNF: 1.0 ±
0.3; AHL: 1.2 ± 0.2) (Figure 2b). GI was slightly improved in
the seeds grown with 0.1 μM AHL than with the control or Fe-
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Figure 4. Profiling of soil microbiome using 16S RNA-based metagenome sequencing after 30 days of plants grown in the presence and absence of NCs
(1 g/kg-soil) and other indicated materials. (a, b) Heap maps indicate bacterial community composition on the basis of (a) phylum and (b) genus
detected in different treatment groups. 16S RNA profiling was performed in the pooled soil sample collected from triplicate experimental pots (n = 3)
of control and each indicated treatment.

CNFs, but the effect was not statistically significant. Next,
seedling growth was calculated in chickpea seeds after 6 days of
growth with/without NCs including Fe-CNFs or AHL alone. A
significant (p < 0.05) increase in the length of seedling was
observed in NC (12.7 ± 2.2 cm)-treated plants with respect to
control (7.8 ± 2.5 cm) and Fe-CNF (9.7 ± 3.0 cm) or AHL (8 ±
1.4 cm). Enhanced seed germination using the NC treatment is
attributed to an increased water intake, as described in our
previous studies.23,30 Similarly, He et al. have shown that the
exposure of graphene oxide (a carbon-based material) at a low
dose in soil enhanced the seed germination of spinach by
triggering water intake in the seeds.37
Nanocomposites Translocate in Chickpea Plants and
Increase the Soil Nitrogen Content. The nanoparticles of
plant micronutrients (e.g., Cu, Zn, or Fe) are shown to be
efficiently translocated in plants, thus enhancing the growth and
yield of the plants, attributed to an increase of micronutrient
bioavailability.38 Liu et al. have shown that spiking the soil with
ZnO nanoparticles influenced the bioavailability of Zn micro-
nutrient in maize plants, which consequently increased plant
316
growth.39 Therefore, we investigated whether Fe was trans-
located in plants through NCs. The captured optical micro-
graphs of the transverse section of root, shoot, and leaf
evidenced the presence of Fe (indicated as black granules) in
the plants grown with NCs or Fe-CNFs alone for 30 days
(Figure S5). However, such granules were absent in the control
or AHL-treated plants. To confirm, SEM and EDS analysis were
performed on shoot sections of the plant. As shown in Figure S6
(SEM micrographs and the corresponding EDS spectra), Fe-
containing carbon nanofibers were distributed in the plant
shoots after 30 days of treatment with NCs and Fe-CNFs.
However, Fe was absent in the control or AHL-grown plants.
Next, we performed AAS measurements on the plants to
determine the actual amounts of Fe (micronutrient) reaching
the plant roots, shoots, and leaves, after growing them for 30
days in NC- or Fe-CNF-spiked soil. As shown in Figure 3a, a
significant (p < 0.05) increase in the Fe content was observed in
the root (0.47 ± 0.01 mg/g), shoot (1.39 ± 0.31 mg/g), and leaf
(0.31 ± 0.02 mg/g) of the plants grown with NCs than in the
plants grown using Fe-CNFs alone (root0.38 ± 0.01 mg/g;
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Figure 5. 16S rRNA sequencing indicates the richness of the
Lactobacillus species in the NC-supplemented soil (1 g/kg-soil) after
30 days. (a, b) Heat map shows the top five most abundant (a)
Lactobacillus, (b) Bacillus species found in control, and different
treatment groups. 16S RNA profiling was performed in the pooled soil
sample collected from triplicate experimental pots (n = 3) of control
and each indicated treatment.
shoot0.49 ± 0.01 mg/g; and leaves0.25 ± 0.01 mg/g) or
control (root0.31 ± 0.02 mg/g; shoot0.43 ± 0.03 mg/g;
and leaves0.21 ± 0.01 mg/g). The maximum content of Fe
was found in the shoot, followed by that in the root of plants.
Interestingly, only NC-grown plants showed a significant (p <
0.05) increase of Fe content in leaves, relative to that in control
and Fe-CNF-grown plants (Figure 3a). In addition, Fe content
in the plant shoots grown with NCs was significantly (p < 0.05)
higher than that of Fe-CNF alone. Furthermore, a mass-balance
calculation of Fe content indicates that each plant receives up to
4 mg of iron after 30 days of treatment with NCs (Supporting
Information Section S1). These results indicate that the
interactions of Fe-CNFs with AHL have a positive impact on
its translocation through the plant vascular system.
Previous studies have shown that AHL can influence the soil
rhizospheric nitrogen mineralization by modulating the micro-
bial community.40 Therefore, we investigated whether NCs
influence the total nitrogen content of rhizospheric soil after 30
days of plant growth. As shown in Figure 3b, relatively higher
nitrogen content was found in soil after 30 days of plant grown
under Fe-CNFs (277.4 kg/hec), AHL (314.1 kg/hec), and
AHL/Fe-CNFs (388.08 kg/hec), stimulus with respect to the
control (110.9 kg/hec). An increased nitrogen content is
measured in soil after treatment with AHL/Fe-CNFs because of
the combined roles of AHL and Fe-CNFs. AHL facilitates the
influx of microbial communities in soil.41 Iron increases the
nitrogenase enzyme activity in microbes, which in turn increases
the biological nitrogen fixation. Nitrogenase is an iron-
containing enzyme in microbes involved in the biological
nitrogen fixation in soil.42 Therefore, supplementing iron in the
form of Fe-CNFs triggers microbially driven nitrogen fixation.
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Figure 6. Abundance of lactobacillus bacteria in the soil after 30 days of
plant growth in the presence and absence of NCs (1 g/kg-soil). The soil
was serially diluted and Lactobacillus sp. was grown in selective media:
(a) optical photomicrographs of Petri dishes show the growth of
Lactobacillus bacteria in the selective MRS medium. (b) Number of
Lactobacillus colonies was quantified in control and different treatment
groups. The inset image indicates the presence of Gram-positive rod-
shaped Bacillus species of bacterium in the soil after 30 days.
The previous studies have also shown that iron can improve the
plant−microbial symbiosis in leguminous plants, which
increases nitrogen fixation and plant growth.43,44
Nanocomposites Improve the Rhizospheric Micro-
biome. The microbial community in the rhizosphere plays a
crucial role in the nitrogen cycle, its availability to the plants, and
mineralization.45 AHL controls the growth of the microbial
community in the rhizosphere, especially Proteobacteria.40 We
performed 16S rRNA metagenome sequencing to understand
the microbial community compositions in soil after 30 days of
plant growth. As shown in Table S1, we could analyze more than
1400 reads, of which more than 1000 reads were classified in
control. On the other hand, more than 5000 reads were analyzed
in the NC-amended rhizosphere soil, of which >3500 reads were
classified. Proteobacteria was the most abundant genus detected
in soil followed by Firmicutes (Figure 4a). In addition, we could
see enrichment in the total number of both bacterial genera
detected in the NC treatment (Proteobacteria − 2336, Firmicutes
− 1098) with respect to control (Proteobacteria − 672,
Firmicutes − 343) or individual treatment to Fe-CNFs
(Proteobacteria − 1456, Firmicutes − 655) and AHL
(Proteobacteria − 1318, Firmicutes − 733). Next, we observed
that Gammaproteobacteria and bacilli were the most abundant
in the top 3% classes detected in soil (Figure S7). Furthermore,
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Figure 7. Growth of chickpea plants after 30 days of supplementation
with NCs (1 g/kg). (a) Plant growth experiments were performed in
pots supplemented with NCs (1 g/kg-soil), Fe-CNFs (1 g/kg-soil),
AHL (0.1 μM), or water only (control). (b−f) Growth and nutritional
quality of plants were measured after harvesting (b) biomass, (c) root
length, (d) shoot length, (e) chlorophyll content, and (f) protein
content. Data presented are mean ± SD (n = 3). Statistical significance
was calculated by applying the one-way ANOVA and Tukey’s post hoc
tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were
considered significant with respect to the control. #p < 0.05, ##p < 0.01,
and ###p < 0.001 indicate significance among individual treatments.
analysis of the bacterial community based on phylum showed
that Lactobacillus was the most enriched phyla in soil, followed
by Bacillus, which were abundant in NC treatment as compared
to the control and individual treatments (Figure 4b).
Interestingly, Lactobacilli are the well-established plant probiotic
microorganisms and biocontrol agents in agriculture.46,47 In
addition, Bacillus spp. (e.g., B. cereus and B. pumilus) are known
for multiple beneficial traits including PGPR and atmospheric
nitrogen-fixing activity.48,49 Furthermore, the analysis of species
distribution within the Lactobacillus and Bacillus genera showed
Lactobacillus reuteri and Bacillus cereus CL1 to be the most
enriched bacterial species in soil (Figure 5a,b). The enrichment
of detected species further improved in the NC-amended soil
with respect to the control and individual treatment groups. To
further validate the 16S rRNA sequencing results, we specifically
cultured Lactobacillus bacteria from the collected soil using
selective culture media and compared their abundances in
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control and treatment groups. As shown in Figure 6, an increase
in the Lactobacillus bacterial colonies was observed in the soil
amended with NCs, as compared to that in control and
individual treatments. The Gram staining of the bacteria from
isolated colonies further confirms the Gram-positive character-
istics of the isolated bacteria.
Nanocomposites Enhance Growth and Nutritional
Quality in Chickpea Plants. To further understand whether
the improved microbiome in NC-treated soil was involved or
not in plant growth and nutrition, we measured the biomass and
nutritional quality of plants after 30 days of growth. As shown in
Figure 7a, we supplemented the chickpea plants with 1 g/kg of
the NC and compared its effect on the plants grown in water
(control) and individual treatments (AHL or Fe-CNF). As
expected, a significant (p < 0.05) improvement is measured in
the fresh biomass of the plants including root and shoot lengths
in the NC-treated plants as compared to the control and
individual treatments. Protein and chlorophyll contents can be
an indicator of plant nutritional quality.23 As shown in Figure
7e,f, a significant (p < 0.05) increase was measured in both total
chlorophyll and protein contents of the plants grown with NCs
as compared to the control and individual treatments. The total
chlorophyll was an additive content of chlorophyll a and b, both
of which significantly (p < 0.05) increased with the NC
treatments. Plants that were grown using Fe-CNF (1 g/kg)- or
AHL (0.1 μM)-amended soil also had significant improvement
in growth (biomass, root-shoot lengths) and nutritional quality
(chlorophyll and protein contents) as compared to control, but
the effects were less pronounced than in the NC-grown plants.
Similar effects were also seen in our previous study performed
using AHL, Fe-CNFs, and NCs in chickpea plants.30 In addition,
a few studies have shown that the other metal- or carbon-based
nanoparticles can trigger plant growth. For example, Wang et al.
reviewed the available data sets on a variety of nanomaterials that
have shown the plant growth promotion activity and concluded
that over a certain range of doses, cerium oxide nanoparticles
(100−200 μg/mL), iron oxide nanoparticles (20−50 μg/mL),
copper nanoparticles (1−50 μg/mL), and zinc oxide nano-
particles (<50 μg/mL) triggered beneficial effects on plants;
however, a dose beyond the indicated range posed deleterious
effects.13 Pandey et al. studied the plant growth effects of carbon-
based nanomaterials in two major bioenergy crops (sorghum
and switchgrass) using graphene and carbon nanotubes.50 The
study showed that the exposure of graphene (200 mg/L) to
switchgrass resulted in a 28% increase in total biomass produced
compared to that in untreated plants. Shekhawat et al. tested the
plant growth-promoting activity of carbon nanoparticles in
Vigna radiata (L.) Wilczek by exposing them in a hydroponic
medium for 96 h.51 The data showed increased plant biomass
including chlorophyll and protein contents after 4 days of
exposure to carbon nanoparticles at 100−150 μM dose.
Nanocomposites Protect Chickpea against Fusarium
Wilt Disease. Manipulation of rhizomicrobiome can suppress
soil-borne plant diseases.6 Therefore, we checked whether the
NC could suppress the pathogen infection of chickpea or not.
Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp.
ciceri is a serious and destructive disease worldwide, which is
translated to 10−90% loss in yield annually.52 We grew the
culture of Fusarium oxysporum f. sp. cicero, and fungal spores
were added to the soil on day 0 together with implanting the
seeds. After 30 days, we could clearly show that control plants
were infected with Fusarium wilt disease, as indicated by curly
and yellowish leaves (Figure 8a). However, plants that were
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Figure 8. NC-treated chickpea plants were resistant to Fusarium wilt disease. (a) Plants were grown in the presence of the indicated fungal pathogen
and 1 g/kg-soil of NCs (except in control) up to 30 days. (b−f) Growth and nutritional quality of plants were measured after harvesting: (b) biomass,
(c) root length, (d) shoot length, (e) chlorophyll content, and (f) protein content. Data presented are mean ± SD (n = 3). Statistical significance was
calculated by applying the one-way ANOVA and Tukey’s post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered
significant with respect to the control. #p < 0.05, ##p < 0.01, and ###p < 0.001 indicate significance among individual treatments.

grown using the NC were free from such symptoms (Figure S8).
Furthermore, we showed that the NC suppressed the incidence
of Fusarium wilt of the chickpea and significantly (p < 0.05)
restored the plant growth (biomass and root-shoot length) and
nutritional quality (chlorophyll and protein contents) in 30 days
of the pot experiments (Figure 8b−f). AHL and Fe-CNFs also
provided some protection but complete protection was only
seen when using the NC. The suppression of fungal patho-
genesis by the NC may be observed because of the influence of
soil microbiome compositions, as discussed above. In line with
319
this finding, Wei et al. have shown that microbiomes of the
plants that survived under pathogenesis were associated with the
specific rare taxa, namely, the highly pathogen-suppressing
Pseudomonas and Bacillus bacteria.4 Li et al. studied the
functional relation of AHL producing quorum-sensing bacteria
in the rhizomicrobiome with the suppression of plant
pathogens.53 Quorum-sensing bacteria belonging to the
Pseudomonas genus showed strong antagonistic activities against
Fusarium oxysporum or Aspergillus flavus, the two main causal
agents of Rehmannia glutinosa root rot disease.53
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A variety of nanoparticles have been shown to trigger the
phytohormone-mediated defense cycle in plants to tackle the
pathogenesis. Cao et al. have shown that foliar application and
seed treatment with organic sulfur nanoparticles (30 and 100 nm
size) suppressed the infection of Fusarium oxysporum f. sp.
Lycopersici in tomatoes, in a concentration- and size-dependent
manner.54 In another study, Ma et al. designed nanoscale
hydroxyapatite (nHA) that suppressed the Fusarium-induced
wilt disease in tomatoes.55 The study showed that the exposure
of plants to nHA triggered the silicic acid-mediated defense
phytohormone pathway, which then controlled the fungal
infection.50 Silicon dioxide nanoparticles have also been shown
to prevent Fusarium wilt disease in watermelon (Citrullus
lanatus) caused by Fusarium oxysporum f. sp. niveum.56 The study
showed that the dissolution of silicon dioxide nanoparticles
mitigated the fungal infection and enhanced the plant growth
and fruit yield in a 2.5 month-long exposure.56 Similarly, El-
Shetehy et al. have shown that the food-graded silicon dioxide
nanoparticles can induce systemic resistance to the bacterial
pathogen in Arabidopsis thaliana by triggering the synthesis of
salicylic acid.57
Overall, our results in the present study indicated that
improving the microbial community composition in plant
habitable soil has a pronounced impact on plant growth, and it
can suppress the pathogen infection in the chickpea plant. Most
importantly, the crops grown under a healthy microbiome are
likely to have a significant effect on human health, considering
that microbes from the plant products (e.g., fruits, salad,
vegetables) can join the human gut microbiome.
■ CONCLUSIONS
The present study has demonstrated that NCs consisting of
short-chain AHL and Fe-CNFs promotes the growth and
nutritional quality of chickpea plants by improving seed
germination, soil N-fixation, biomass production, and chlor-
ophyll and protein contents. Furthermore, the results show that
the NC treatment of soil protects the chickpea plants from one
of the most common fungal pathogens, viz. Fusarium oxysporum
f. sp. cicero, which causes Fusarium wilt disease. By applying the
metagenome-based 16S RNAseq approach, we have shown that
the NC (Fe-CNF/AHL) treatment in the soil−plant system for
30 days potentially improves the rhizomicrobiome composition,
which promotes plant growth and suppresses Fusarium wilt
disease. The findings in this study have great implications in the
field of sustainable agriculture. It is likely that the developed
NCs can play a vital role in reviving the microbiome of plant
habitable soil, which is suppressed or under suppression because
of excessive use of chemical fertilizers.
■
ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsagscitech.1c00217.
Total analyzed and classified/unclassified reads of the
control and treatment group (Table S1);thermal stability
of soil determined by thermogravimetric analysis over
25−100 °C (Figure S1); texture analysis of soil used in
plant growth experiments (Figure S2); elemental content
of the soil used in plant growth experiments (Figure S3);
physiochemical characteristics of NCs (Figure S4);
uptake and translocation of NCs in plants after 30 days
(Figure S5); uptake and translocation of NCs (1 g/kg-
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soil) in plants after 30 days (Figure S6); abundance (top
3%) of the bacterial class detected in the soil after 30 days
of growth (Figure S7); and optical micrographs of plants
after 30 days (Figure S8) (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
Nishith Verma − Center for Environmental Science and
Engineering and Department of Chemical Engineering, Indian
Institute of Technology Kanpur, Kanpur 208016, India;
orcid.org/0000-0002-4599-6468; Phone: +91 512 259
6767; Email: vermanishith@gmail.com, nishith@iitk.ac.in
Govind Gupta − Center for Environmental Science and
Engineering, Indian Institute of Technology Kanpur, Kanpur
208016, India; Present Address: Division of Molecular
Toxicology, Institute of Environmental Medicine,
Karolinska Institutet, Nobels väg 13, Stockholm S-171 77,
Sweden (G.G.); Phone: +46-8524 877;
Email: govindgupta.iitk@gmail.com
Author
Komal Pandey − Center for Environmental Science and
Engineering, Indian Institute of Technology Kanpur, Kanpur
208016, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsagscitech.1c00217
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors acknowledge the financial support received from
the Council of Scientific and Industrial Research (CSIR) (New
Delhi, India) in the form of a research grant (Grant No. 9
2IJ4803)/14). The authors acknowledge technical support from
the Triyat Genomics Pvt. Ltd. (Nagpur, India) for the 16S RNA
metagenome sequencing and analysis.
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