Scientia Horticulturae 249 (2019) 374–382

Contents lists available at ScienceDirect

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

Effects of graphene oxide and zinc oxide nanoparticles on growth, T

chlorophyll, carotenoids, proline contents and diseases of carrot
Zaki A. Siddiquia, , Aiman Parveena, Lukman Ahmada, Abeer Hashemb
⁎

a
Department of Botany, Aligarh Muslim University, Aligarh, 202002, India
b
Botany and Microbiology Department, Faculty of Science, King Saud University, Riyadh, 11451, Saudi Arabia

ARTICLE INFO ABSTRACT

Keywords: The effects of graphene oxide (GO) and zinc oxide (ZnO) nanoparticles (NPs) at two concentrations, i.e.,
Alternaria 0.05 mg ml−1 and 0.10 mg ml−1 were observed on Pectobacterium carotovorum, Xanthomonas campestris pv.
Daucus carotae, Meloidogyne javanica, Alternaria dauci and Fusarium solani on the growth of carrot (Daucus carota).
Fusarium Specific physiological variables studied include chlorophyll, carotenoid and proline contents. Inoculation of
Meloidogyne
plants with P. carotovorum, X. campestris pv. carotae, M. javanica, A. dauci and F. solani reduced plant growth and
Pectobacterium
Xanthomonas
chlorophyll and carotenoid contents but increased proline contents. Application of GO or ZnO NPs to plants with
or without pathogens caused significant increases in plant growth, chlorophyll, carotenoid and proline contents.
ZnO NPs were more effective than GO NPs in increasing plant growth, chlorophyll, carotenoid and proline
contents. The 0.10 mg ml−1 concentration of both NPs was more effective in increasing plant growth, chlor-
ophyll, carotenoid and proline contents than the 0.05 mg ml−1 concentration. Galling and multiplication of M.
javanica was reduced by NPs application. ZnO NPs at concentration, 0.10 mg ml−1 caused highest reduction in
galling and nematode multiplication and GO NPs at 0.05 mg ml−1 the least. Soft rot, bacterial leaf blight, leaf
spot and root rot indices caused by P. carotovorum, X. campestris pv. carotae, A. dauci and F. solani respectively
were 4. Disease indices were reduced to 2 when 0.05 and 0.10 mg ml−1 GO and 0.05 mg ml−1 ZnO were sprayed
onto plants with test pathogens. Indices were highly reduced to 1 when 0.10 mg ml−1 ZnO was sprayed onto
plants with test pathogens.

1. Introduction
Carrot (Daucus carota L.) is herbaceous root vegetable of the
Apiaceae family. It is one of the popular vegetables and has very im-
portant nutritional value (Al-Harbi et al., 1997). It is being increasingly
consumed mainly due to its pleasant flavour and perceived health
benefits related to vitamins, minerals and fibers. Carrot is an excellent
source of iron, calcium, phosphorus, folic acid and vitamin B and rich in
sugar content (Yawalkar, 1992). It is used as salad and as cooked ve-
getable in soups, stews, curries, etc. and also used for the preparation of
pickles, jam, and sweet dishes (Kabir et al., 2000).
Carrot is affected by various plant pathogens. Important diseases of
carrot include; bacterial soft-rot by Pectobacterium carotovorum, bac-
terial leaf blight by Xanthomonas campestris pv. carotae, root-knot by
Meloidogyne spp., fungal leaf blight by Alternaria dauci, and rot by
Fusarium sp. (Nesha and Siddiqui, 2013; Nunez and Davis, 2016).
Pectobacterium carotovorum (syn. Erwinia carotovora) is a species of
gram-negative, facultative anaerobic, rod-shaped bacteria (Whitehead
et al., 2002). It causes soft-rot on carrot, and the disease is characterized
by extensive plant tissue maceration by a variety of secreted enzymes.
Xanthomonas campestris pv. carotae causes leaf blight on carrot. The
pathogen can cause lesions and blight of flower umbels and flower
stalks; a yellow exudate can ooze from such lesions. The presence of
water is necessary for infection to occur, the pathogen reproduces most
rapidly under warm (25 to 30 °C) and wet conditions (Kendrick, 1934;
Pfleger et al., 1974). Leaf spot of carrot caused by Alternaria dauci is one
of the significant foliar diseases. Symptoms of the infection are spotting
on leaves, stems, flowers and fruits and damping-off in seedlings caused
by lesions encircling the roots (Agrios, 2005). The lesions surrounding
the infected area exhibit chlorosis as disease progresses (Agrios, 2005).
Fusarium sp. can cause a spongy rot of carrot roots. The presence of
shallow, scabby spots on the taproot that extend into lesions and later
into rotting symptoms of the disease. Meloidogyne species have great
economic importance; can cause severe damage to several crop plants.
Meloidogyne incognita and M. javanica are widely distributed around the
world (Brand et al., 2010). Among Meloidogyne species, M. javanica is

⁎
Corresponding author.
E-mail addresses: zaki_63@rediffmail.com, zaki_63@yahoo.co.in (Z.A. Siddiqui).

https://doi.org/10.1016/j.scienta.2019.01.054
Received 3 October 2017; Received in revised form 27 January 2019; Accepted 28 January 2019
Available online 12 February 2019
0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Z.A. Siddiqui et al.
one of the most common species found in carrot fields of tropical and
subtropical regions (Peterson and Simon, 1986; Stein and Nothnagel,
1995). Meloidogyne spp. induce root galls and giant cells formation
impair water and nutrient uptake to the shoots (Lordello, 1984).
Nanotechnology offers green and eco-friendly alternatives for plant
disease management (Alghuthaymi et al., 2015). Significant develop-
ment in nanomaterials synthesis has attracted researcher’s attention
towards the management of plant diseases (Ocsoy et al., 2013). Gra-
phene has a large theoretical specific surface area of 2630 m2 /g
(Bitounis et al., 2013; osSantos et al., 2014), high intrinsic mobility of
200,000 cm2 /v/sec, thermal conductivity of ˜5000/Wm/K (Shao et al.,
2010), and optical transmittance of ˜97.7%. Graphene oxide (GO), the
functionalized graphene with oxygen-containing chemical groups, has
superior properties such as large surface area, mechanical stability,
tunable electrical and optical properties (Li et al., 2015). Moreover, the
surface functional groups of hydroxyl, epoxy and carboxyl make GO an
excellent candidate in coordinating with other materials or molecules.
The expanded structural diversity and improved overall properties, GO
hold great promise for versatile applications (Li et al., 2015). However,
the antibacterial activities of individual graphene nano- sheets or GO
are limited (Nanda et al., 2015). Zinc oxide nanoparticles (ZnO NPs)
have remarkable optical, physical, and antimicrobial properties and
therefore, have great potential to enhance agriculture (Sabir et al.,
2014). Zinc oxide nanoscale treatments promote seed germination,
seedling vigor, and plant growth and are also effective in increasing
stem and root growth in peanuts (Prasad et al., 2012). Moreover, these
NPs constitute an effective antimicrobial agent against pathogenic mi-
croorganisms (Sabir et al., 2014). The detected active oxygen species
generated by these metal oxide particles may be responsible for their
antimicrobial activity. ZnO NPs are by far less toxic to plants and plant-
beneficial soil bacteria (Gajjar et al., 2009; Stampoulis et al., 2009;
Dimkpa et al., 2012).
During the course of survey of carrot fields of Aligarh district of
U.P., we found frequent occurrence of Pectobacterium carotovorum
(Jones) Waldee, Xanthomonas campestris pv. carotae (Pammel) Dowson,
Meloidogyne javanica (Treub) Chitwood, Alternaria dauci (J.G. Kühn)
J.W. Groves and Skolko and Fusarium solani (Martius) Sacc. Plants in-
fected with above mentioned pathogens were poor in growth with se-
vere disease symptoms. Therefore, these pathogens are highly de-
structive and major constraints in the successful cultivation of carrot.
Both nanoparticles i.e. GO and ZnO were applied on the foliage
parts of carrot to avoid direct contact with the soil ecosystem. The aim
of this study was to explore the use of GO and ZnO NPs for the man-
agement of Pectobacterium carotovorum, Xanthomonas campestris pv.
carotae, Meloidogyne javanica, Alternaria dauci and Fusarium solani on
carrot by minimizing risk of NPs toxicity. Effect of both NPs on plant
growth and physiological responses were also investigated.
2. Materials and methods
Root and soil samples were collected from carrot fields of Aligarh
district, U.P., India. These samples were collected in polythene bags and
stored in a refrigerator at 4 °C until processing began. The samples were
examined for the presence of the root-knot nematode Meloidogyne spp.,
fungi and plant pathogenic bacteria.
2.1. Isolation and identification of root-knot nematode M. javanica
Carrot roots were examined for root-knot symptoms. Root galls
were dissected to take out the females of root-knot nematodes.
Identification of Meloidogyne sp. was made on the basis of perineal
patterns (Taylor and Sasser, 1978). Soil samples were also processed for
the nematodes isolation by Cobb’s sieving and decanting technique
followed by a Baermann funnel (Southey, 1986). Identification of root-
knot juveniles was also made from the nematode suspension.
375
Scientia Horticulturae 249 (2019) 374–382
2.1.1. Isolation of fungi from infected carrot
Fungal disease symptoms on both shoots and roots were transferred
to sterilized Petri plates containing sterilized distilled water and gently
freed of soil particles. These parts were transferred to other Petri plates
and the process was repeated until all adhering soil particles were re-
moved. Later, these infected parts were cut into approximately 5 mm
pieces and transferred to a Petri dish containing 0.1% sodium hypo-
chlorite (NaOCl) solution. After one minute the pieces were washed at
least thrice in distilled water and dried on filter paper. Five of these
pieces were then plated in Petri plates containing PDA (Potato dextrose
agar) using sterilized forceps under aseptic conditions. Petri plates were
incubated at 25 ± 2 °C for 10 days. The fungus that developed from
infected root and shoot pieces were examined and identified. The
identification of Fusarium species is mainly based on distinctive char-
acters of the shapes and sizes of macro- and microconidia, presence and
absence of chlamydospores as well as colony appearances, pigmenta-
tions and growth rates on agar media (Leslie and Summerell, 2006). For
identification of Alternaria species, culture morphology, growth rate
and conidial morphology were observed from 12 to 15 day-old cultures
grown on PDA and PCA (Simmons, 2007). The shape, length and width
of 50 conidia for each isolate were determined and mean length and
width were calculated. In addition, the number of transepta per con-
idium and the production of conidia in catenate arrangement was de-
termined (Tulek and Dolar,2015). On confirmation of identity as Al-
ternaria dauci and Fusarium solani, pure cultures of these fungi were
prepared. The pure cultures were stored at 5 °C until used. The com-
position of PDA medium was as follows:
Potato infusion form 200 g
Dextrose 20 g
Agar agar 15 g
Distilled water 1000 ml
2.1.2. Isolation of bacteria from infected carrot
Collected carrot plants with bacterial disease symptoms were used
for the isolation of the bacteria. Plant parts having rot or blight
symptoms were transferred to sterilized petri plates containing ster-
ilized distilled water and gently freed of soil particles. Sterilization in
NaOCl was done as described above. Five of these sterilized pieces were
then plated in each of the Petri plates containing nutrient agar medium
with the help of sterilized forceps under aseptic condition. Petri plates
were then incubated at 30 + 2 °C for 24–48 h. The bacteria that de-
veloped from infected pieces were examined and identified. P. car-
otovorum characterized positive in pectolytic activity was tested for the
conventional biochemical and physiological tests including Gram re-
action, fermentative metabolism, oxidase and catalase activity.
Production of reducing substances from sucrose, indole production
from tryptophan, were also studied. Bacterium was also checked for
production of phosphatase and amylase, sensitivity to erythromycin
Schaad et al. (2001), ability to grow at 37 °C in nutrient broth and
growth in 5% sodium chloride on nutrient agar at 28 °C. In addition, the
assimilation of carbon sources were tested on the basal medium (Ayers
et al., 1919) supplemented with 1% carbohydrates including lactose,
glucose, fructose and mannitol. For Identification of X. campestris pv.
carotae, isolate was grown on MDS medium. Colonies of X. campestris
pv. carotae were typically straw yellow, glistering, round, smooth,
convex with entire margins and 3–5 mm in diameter (Kuan and
Minsavage, 1985). The isolate was further tested biochemically for
casein hydrolysis, anaerobic growth, urease production, catalase pro-
duction, aesculin hydrolysis, starch hydrolysis, production of fluor-
escent pigment on King’s B medium and arginine dihydrolase activity
(Dye, 1980). On confirmation of identity as Pectobacterium carotovorum
and Xanthomonas campestris pv. carotae pure cultures of these bacteria
were prepared and stored at 5 °C. The composition of nutrient agar
medium was as follows:
Z.A. Siddiqui et al.
Beef extract 3.0 g
Peptone 5.0 g
Agar agar 15.0 g
Distilled Water 1.0 liter
2.1.3. Preparation of graphene oxide (GO NPs) solution
Graphene oxide (GO), form dispersion in H2O, concentration 2 mg /
ml, refractive index n20/D 1.333, density 0.981 g / mL at 25 °C (GO,
Aldrich product no. 763,705). GO with large surface area, high che-
mical stability, good charge carrier properties. Chloride free (purified
by dialysis) monolayer sheet, mean sheet diameter: 22 μm, 90% below
50 μm by laser diffraction. Graphite oxide is easily dispersed in water,
where it breaks up into macroscopic atomically thin flakes, leading to
solutions/suspensions of single layer GO. A typical GO solution has a
brownish appearance, whose transparency and 'strength' is tuned by
varying the solute concentration. Solutions of GO were prepared by
dissolving 25.0 ml and 50.0 ml solutions of GO NPs in one litre distilled
water. Since GO is a solution of 0.2% (Sigma Aldrich) after dilution
(25.0 ml and 50.0 ml solutions in one litre distilled water separately)
the final concentrations of solution became 0.05 mg ml−1 and
0.10 mg ml−1 for the foliar spray respectively. Ten ml suspension of GO
NPs was used as foliar spray per pot / per plant.
2.1.4. Preparation of zinc oxide nanoparticles solution
Zinc oxide, dispersion nanoparticles (ZnO NPs), < 100 nm particle
size (TEM), ≤40 nm avg. particle size (APS), 20 wt. % in H2O, pH
7.5 ± 1.5 was obtained from Sigma-Aldrich (Product No. 721077-
100 G). Appearance or color of ZnO is white. X-Ray diffraction con-
firms to structure complexometric titration 79.1–81.5 % Zn. Percentage
Zn size < 100 nm while average particle size 15–25 nm while specific
surface area (m2/g). Solution of ZnO NPs was prepared by dissolving
0.25 and 0.50 ml ZnO NPs in one litre distilled water separately for the
foliar spray. Since ZnO NPs is already a solution of 20% (Sigma Aldrich)
after dilution (0.25 ml and 0.50 ml in one litre DDW separately), ZnO
NPs concentration became 0.005% and 0.01% respectively
(i.e.0.05 mg/ ml and 0.10 mg / ml). Ten ml suspension of ZnO NPs was
used as foliar spray per pot / per plant.
2.1.5. Effect of GO and ZnO NPs on hatching and mortality of root knot
nematodes
The effect of GO and ZnO NPs (0.05 mg ml−1 and 0.10 mg ml−1
concentrations) were observed on the hatching and mortality of M.
javanica in small Petri plates at 25 ± 1 °C. Twenty egg masses of almost
similar size were handpicked with sterilized forceps from the roots of
eggplant (Solanum melongena L.) and were placed for hatching in 20 ml
suspensions of GO / ZnO NPs of 0.05 mg ml−1 and 0.10 mg ml-1 con-
centrations. For control, twenty egg masses were placed in 20 ml double
distilled water. Each set was replicated five times and observed under
stereomicroscope.
2.1.6. Effect of GO and ZnO NPs on the fungi
The antifungal activity of GO and ZnO NPs was determined by
adding 10 ml GO / ZnO NPs (0.05 mg ml-1 and 0.10 mg ml-1 con-
centrations) into 100 ml autoclaved PDA separately just before pouring
in Petri plates. A. dauci and F. solani were inoculated on the plates se-
parately containing 0.05 mg ml-1 and 0.10 mg ml-1 concentration of GO
/ ZnO NPs in PDA. Each set was replicated five times. The plates were
incubated at 25 ± 2 °C for 10 days. Antifungal activity of GO and ZnO
NPs was determined by measuring the growth of fungal colonies 10
days after inoculation. Percent inhibition was calculated with control.
2.1.7. Effect of GO and ZnO NPs on the bacteria
Pectobacterium carotovorum and X. campestris pv. carotae were in-
oculated under aseptic condition on the nutrient agar plates separately.
The antibacterial activity of GO and ZnO NPs was determined by
376
Scientia Horticulturae 249 (2019) 374–382
placing aseptically paper discs of 0.7 mm diameter dipped in GO / ZnO
NPs (0.05 mg ml-1 or 0.10 mg ml-1 concentration) separately into nu-
trient agar plates Each set was replicated five times. The plates were
incubated at 30 ± 2 °C and observed every 24–48 h. Antibacterial ac-
tivity of GO and ZnO NPs was determined by measuring the inhibition
zone 48 h after inoculation.
2.2. Preparation and sterilization of soil mixture
Sandy loam soil is collected from a field belonging to the
Department of Botany, A.M.U., Aligarh and passed through a 10 mesh
sieve. The soil, river sand and organic manure were mixed in the ration
3:1:1 and 15cm diameter clay pots were each filled with 1 kg of the
mixture. A little water was poured into each pot to just wet the soil
surface before sterilization at 137.9 kPa for 20 min. Sterilized pots were
allowed to cool at room temperature before use.
2.3. Raising and maintenance of test plants
Carrot (Daucus carota L.) seeds Cultivar Laali were surface sterilized
with 0.1% sodium hypochlorite (NaOCl) for 2 min and rinse three times
with sterile water. Three seeds were sown in steam sterilized soil in
15 cm diameter clay pots and one week after germination thinning was
done to maintain single seedling per pot. Seedlings were subjected to
the treatments listed in Table 3. Un-inoculated plants served as a con-
trol and plants were kept in a glasshouse at 20 ± 2 °C. Pots were ar-
ranged in a randomize block design and each treatment was replicated
5 times. Pots were watered as needed and experiment was terminated
90 days after inoculation.
2.4. Inocula of fungi
For obtaining sufficient inocula, A. dauci and F. solani were sepa-
rately cultured on Richard’s liquid medium having following compo-
sition:
Potassium nitrate 10.0 gm
Potassium dihydrogen phosphate 05.0 gm
Magnesium sulphate 02.5 gm
Ferric chloride 0.02 gm
Sucrose 50.0 gm
Distilled water 1000 ml
The medium was prepared and filtered through muslin cloth,
poured 80 ml liquid medium in each 250 ml Erlenmeyer flasks. These
flasks were sterilized in an autoclave at 103.4 kPa for 15 min. Alternaria
dauci and F. solani were separately inoculated in each flask with the
help of inoculation needle. Inoculated flasks were incubated at
25 ± 1 °C for about 15 days to allow sufficient growth of each fungus.
Mycelia mats of each fungus were collected on blotting papers sepa-
rately to absorb excess of water and nutrients. The inoculum of each
fungus was prepared separately by mixing 50 g mycelium in 500 ml
distilled water and blending it for 30 s in Waring blender. Ten ml of this
suspension contain 1 g fungus was used as inoculum. Pure cultures of A.
dauci and F. solani were continuously maintained on PDA by re-in-
oculation after every 15 days.
2.5. Inocula of bacteria
For obtaining sufficient inocula, P. carotovorum and X. campestris pv.
carotae were separately cultured on Nutrient agar medium. Medium
was poured in sterilized petri-plates and these plates were later streaked
separately with a pure colony of P. carotovorum / X. campestris pv.
carotae and incubated at 30 ± 1 °C for 24 h. Single colonies from a 24-
h-old pure culture of P. carotovorum / X. campestris pv. carotae were
Z.A. Siddiqui et al.
inoculated separately into nutrient broth flasks and incubated at
30 ± 1 °C for 72 h. Cell density was determined following Sharma
(2001) and measured 1.2 × 105 colony-forming units (CFU) / ml.
2.6. Preparation of nematode inoculum
Meloidogyne javanica was collected from the lentil roots and multi-
plied on the roots of egg plants (Solanum melongena L.) using single egg
mass. Large numbers of egg masses from heavily infected eggplant roots
were hand-picked with the help of sterilized forceps from the previously
maintained pure culture of M. javanica. The egg masses were washed
with distilled water and placed in a small sieve (9 cm diameter with 1-
mm pore size) containing crossed layers of tissue paper. The sieve was
placed in a Petri plate containing distilled water deep enough to contact
the egg masses. A number of these assemblies were kept in an incubator
running at 25 ± 1 °C in order to obtain the required number of second-
stage juveniles for inoculation. The hatched second-stage juveniles were
collected from the Petri plates every 24 h, fresh water was added, and
the process was repeated. For counting nematode juveniles, an average
of 5 counts was made to determine the density of nematodes in the
suspension. The volume of nematode suspension was so adjusted that
each ml may contain 200 ± 5 nematodes. Ten ml of this suspension
(i.e. 2000 freshly hatched juveniles) was added to each pot around a
carrot seedling.
2.7. Inoculation technique
For inoculation of M. javanica, fungi and bacteria, soil around the
roots was carefully removed a side without damaging the roots. The
inoculum suspensions were poured around the roots and the soil was
replaced. In control treatment, water was added in equal volume to the
suspension. The treatments applied are shown in Table 3. There were 6
treatments comprising of (1) Control; (2) M. javanica ; (3) P. car-
otovorum ;(4) X. campestris pv. carotae ; (5) A. dauci ;(6) F. solani. These
6 treatments were tested with (I) Control; (II) GO NPs 0.05 mg ml-1 (III)
GO NPs 0.10 mg ml−1 (IV) ZnO NPs 0.05 mg ml-1 (V) ZnO NPs
0.10 mg ml-1. Each treatment was replicated 5 times (6 × 5×5 = 150
pots). Experiment was conducted in 2015 and repeated in 2016. Data of
both the experiments were almost similar, paper presents the pooled
data of both years.
2.8. Observations
The plants were harvested 90 days after inoculation. Data on plant
length, galls and root-rot, leaf spot and blight indices were recorded.
The length of plant was recorded in centimeter from the top of the first
leaf to the end of the root. Excess water was removed by blotting before
weighing the plant. The plants were cut with knife above the base of the
root emergence zone to separate shoot and root. Shoot and root were
kept in envelope at 60 °C for 4 days before weighing for dry weight
determination. A 250 g subsample of well-mixed soil from each treat-
ment was processed by Cobb’s sieving and decanting technique fol-
lowed by Baermann funnel extraction (Southey, 1986). Nematode
suspension was collected after 24 h and the numbers of nematodes were
counted in five aliquots of 1 ml of suspension from each sample. The
means of five counts were used to calculate the population of nema-
todes per kg soil. To estimate the number of juveniles, eggs and females
inside the roots, 1 g subsample of roots was macerated in a Waring
blender and counts were made from the suspension thus obtained.
Numbers of nematodes present in roots were calculated by multiplying
the number of nematodes present in 1 g of root by the total weight of
root. Soft rot, leaf blight, leaf spot and root-rot indices were determined
by scoring the severity of disease on a scale ranging from 0 (no disease)
to 5 (severe disease).
377
Scientia Horticulturae 249 (2019) 374–382
2.9. Estimation of total chlorophyll and carotenoids contents
The chlorophyll and carotenoid content in the fresh leaf was esti-
mated following the method of Mackinney (1941). One g of freshly cut
leaves was ground to fine pulp using a mortar and pestle after pouring
20 cm³ of 80% acetone. The mixture was centrifuged at 5000 rpm for
5 min. The supernatant was collected in 100 cm³ volumetric flask. The
residue was washed three times, using 80% acetone. Each washing was
collected in the same volumetric flask and volume was made up to
mark, using 80% acetone. The absorbance was read at 645 and 663 nm
for chlorophyll and 480 and 510 nm for carotenoid against the blank
(80% acetone) on spectrophotometer (Shimadzu UV-1700, Tokyo,
Japan).
2.10. Estimation of proline content
The proline content in fresh leaves was estimated following the
method of Bates et al. (1973). Briefly fresh leaf sample (300 mg) was
homogenized in 3 ml of 3 percent sulphosalicylic acid and the homo-
genate filtered and filtrate was reacted with 1 ml each of ninhydrin and
glacial acetic acid for one hour in a test tube placed in a warm water
bath at 100 °C. Finally the sample was transferred to ice bath and
mixture was extracted with toluene and was read at 520 nm using L-
proline as a standard.
2.11. Statistical analysis
Two way ANOVA (NPs × Pathogens) was used to test significance
(p ≤ 0.05) of data, i.e., plant length, plant fresh weight, shoot and root
dry weight, chlorophyll, carotenoid and proline contents. Least sig-
nificant differences were calculated for NPs, Pathogens and their in-
teractions. Duncan’s new multiple range test was employed to show
significant differences between the treatments. Sigma Plot™ was used
for preparation of graphs of nematode population and number of galls
per root system. Error bars indicate the standard error.
3. Results
3.1. Effects of GO and ZnO NPs on bacteria, fungi and nematodes
GO and ZnO NPs had an inhibitory effect on growth of P. car-
otovorum, and X. campestris pv. carotae (Table 1). ZnO NPs had a greater
inhibitory effect than by GO NPs, resulting in the formation of broad
inhibition zones. Inhibition zone formation was also dependent on
concentration of NPs in the medium. An inhibition zone of 0.72 mm
formed around the 0.10 mg ml-1 ZnO NP discs on plates inoculated with
P. carotovorum and 0.62 mm around X. campestris pv. carotae (Table 1).
However, the 0.05 mg ml-1 ZnO disc resulted in a 0.36 mm inhibition
zone against P. carotovorum and 0.29 mm inhibition zone with X.
campestris pv. carotae. Application of 0.10 mg ml-1 GO NPs discs re-
sulted in a 0.53 mm inhibition zone with P. carotovorum, and 0.41 mm
with X. campestris pv. carotae (Table 1). However, the 0.05 mg ml-1 GO
disc resulted in a 0.28 mm inhibition zone with P. carotovorum, and a
0.20 mm inhibition zone with X. campestris pv. carotae (Table 1).
GO and ZnO NPs also had an inhibitory effect on growth of A. dauci,
and F. solani (Table 1). ZnO NPs had a greater inhibitory effect than
caused by GO NPs, resulting in the higher antifungal activity. Reduction
in fungus growth was also dependent on concentration of NPs in the
medium. Reduction in the growth of A. dauci was 52.5% in 0.10 mg
ml-1 ZnO NP concentration (10 ml ZnO NPs added in 100 ml PDA) and
47.4% reduction in the growth of F. solani (Table 1). However, the
0.05 mg ml−1 ZnO resulted in 24.7% reduction in the growth of A. dauci
and 22.4% reduction in the growth of F. solani. Application of
0.10 mg ml-1 GO NPs resulted in a 36.2% reduction in the growth of A.
dauci, and 33.7% reduction in the growth of F. solani (Table 1). How-
ever, the 0.05 mg ml-1 GO resulted in a 21.4% reduction in the growth
Z.A. Siddiqui et al. Scientia Horticulturae 249 (2019) 374–382

Table 1 Similarly, 82.92 and 95.83% mortality was observed in ZnO 0.05 mg ml-1
Effects of GO and ZnO NPs on the fungi and bacteria in culture medium and and 0.10 mg ml-1 concentrations in 48 h (Table 1).
effects of NPs on the hatching and mortality of M. javanica.
Treatments Inhibition zone formation against bacteria in mm 3.2. Greenhouse experiment

GO 0.05 mg/ ml P. carotovorum 0.28 f 3.2.1. Effect on plant growth, chlorophyll, carotenoid and proline contents
X. campestris 0.20 g
Inoculation of F. solani caused highest reduction in plant growth
GO 0.10 mg/ ml P. carotovorum 0.53 c
X. campestris 0.41d followed by A. dauci and P. carotovorum (Table 2). M. javanica caused
ZnO 0.05 mg/ ml P. carotovorum 0.36 e least reduction in plant growth and statistically similar to that caused
X. campestris 0.29f by X. compestris pv. carotae. Foliar spray of GO / ZnO NPs at two con-
ZnO 0.10 mg/ ml P. carotovorum 0.72 a centrations, i.e., 0.05 and 0.10 mg ml−1 resulted in a significant in-
X. campestris 0.62 b
crease in plant growth over control (Table 2). ZnO NPs at 0.10 mg ml-1
Treatments Antifungal activity of NPs in percentage
resulted in a greater increase in plant growth followed by ZnO NPs at
0.05 mg ml−1 and 0.10 mg ml−1 GO NPs. Spray with 0.05 mg ml−1 GO
GO 0.05 mg /ml A. dauci 21.4f NPs was least effective (Table 2).
F. solani 19.2g Foliar spray of GO / -ZnO NPs at both concentrations caused a
GO 0.10 mg /ml A. dauci 36.2c
significant increase in chlorophyll, carotenoid and proline contents
F. solani 33.7d
ZnO 0.05 mg /ml A.dauci 24.7e (Table 2). Spray of 0.10 mg ml−1 ZnO NPs caused a greater increase in
F. solani 22.4f chlorophyll, carotenoid, and proline contents followed by spray of
ZnO 0.10 mg /ml A. dauci 52.5a 0.05 mg ml−1 ZnO NPs. Spray of 0.05 mg ml-1 ZnO NPs caused statis-
F. solani 47.4b tically similar chlorophyll, carotenoid and proline contents as caused by
Treatments Hatching of M. javanica J2 after Mortality of J2 after spray of 0.10 mg ml-1 GO NPs while spray with 0.05 mg ml−1 GO NPs
48 hours 48 hours was least effective. Inoculation of A. dauci caused a greater reduction in
chlorophyll and carotenoid contents but statistically similar to that
Distilled water 211a 8e
caused by F. solani. Inoculation of P. carotovorum caused statistically
GO 0.05 mg/ ml 129b 83a
GO 0.10 mg/ ml 68d 59c similar reduction in chlorophyll and carotenoid contents as caused by
ZnO 0.05 mg/ ml 82c 68b X. compestris pv. carotae. Inoculation of M. javanica caused least re-
ZnO 0.10 mg/ ml 24e 23d duction in chlorophyll and carotenoid contents (Table 2).
*Values within column and within one type of pathogens i.e. fungi/ bacteria Inoculation of P. carotovorum, X. campestris pv. carotae, M. javanica, A.
/nematode followed by different letters are significantly different at P = 0. 05 dauci and F. solani caused soft rot, leaf blight, root knot, leaf spot and root
based on Duncan’s new multiple range test. rot symptoms respectively on carrot (Fig. 1). Spray of 0.10 mg ml-1 GO /
ZnO NPs in both concentrations to plants without pathogens resulted in
significant increases in shoot and root dry weight over uninoculated
of A. dauci, and 19.2% reduction in the growth of F. solani (Table 1). control (Table 3). Similarly, application of GO/ ZnO NPs to plants with P.
Effects of GO and ZnO NPs (0.05 mg ml−1 and 0.10 mg ml-1 con- carotovorum, X. campestris pv. carotae, M. javanica, A. dauci and F. solani
centrations) were studied on the hatching and mortality of M. javanica caused a significant increase in growth over plants inoculated with test
(Table 1). GO and ZnO NPs in both concentrations had inhibitory effect pathogens only. Application of 0.10 mg ml-1 ZnO NPs to plants with P.
on hatching of M. javanica. ZnO NPs was more effective in reducing carotovorum, X. campestris pv. carotae, M. javanica, A. dauci and F. solani
hatching than caused by GO NPs. GO NPs caused 38.86 and 67.77% resulted in a greater increase in shoot and root dry weight than for plants
inhibition in hatching over control in 0.05 mg ml-1 and 0.10 mg ml−1 sprayed with 0.05 mg ml-1 ZnO, 0.10 mg ml-1 GO or 0.05 mg ml-1 GO
concentrations respectively. Similarly, ZnO NPs caused 61.13 and NPs. Spray of 0.05 mg ml-1 ZnO NPS to plants with P. carotovorum, X.
88.63% inhibition in hatching over control in 0.05 mg ml−1 and campestris pv. carotae, M. javanica, A. dauci and F. solani resulted in sta-
0.10 mg ml−1 concentrations respectively. Mortality of M. javanica 2nd tistically similar plant growth as was caused by spray of 0.10 mg ml-1 GO
stage juveniles was 3.79% in double distilled water in 48 h. Mortality of NPs. Spray with 0.05 mg ml−1 GO NPs to plants with test pathogens was
M. javanica 2nd stage juveniles in GO was found 64.34 and 86.76% re- least effective in increasing plant growth (Table 3).
spectively in 0.05 mg ml−1 and 0.10 mg ml−1 concentrations in 48 h. Spray of GO 0.10 mg ml-1 or ZnO NPs at both concentrations to

Table 2
Effect of GO and ZnO NPs on the growth, chlorophyll, carotenoid and proline contents of carrot in pathogen inoculated and Uninoculated plants.
Treatments Plant length (cm) Plant fresh weight (g) Dry weight(g) Total Chloro- Carotenoid content (mg/g FW) Proline content (μmolg−1 FW)
phyll content (mg/g FW)
Shoot Root

Control 73.51a 64.15a 5.36a 7.41a 0.336a 0.0458a 0.0814f

M. javanica 63.43b 58.46b 4.31b 6.33b 0.301b 0.0421b 0.1014e
P. carotovorum 61.87c 54.96d 3.99c 6.00d 0.291c 0.0402cd 0.1072c
X.campestris 63.27b 57.10c 4.46b 6.33b 0.293c 0.0413bc 0.1044d
A.dauci 59.50d 53.20e 3.73d 6.67c 0.271d 0.0382e 0.1166a
F. solani 56.94e 49.69f 3.50e 5.45e 0.279d 0.0392de 0.1126b
Least Square Mean 63.08 56.26 4.22 6.20 0.295 0.0411 0.1039

Control 56.27e 49.82e 2.90e 4.77e 0.241d 0.0357d 0.0976d

GO 0.05 mg /ml 62.57d 55.31d 3.70d 5.64d 0.276c 0.0392c 0.1024c
GO 0.10 mg /ml 64.38c 57.73c 4.36c 6.50c 0.307b 0.0423b 0.1050b
ZnO 0.05 mg /ml 65.17b 58.27b 4.57b 6.69b 0.305b 0.0427b 0.1055b
ZnO 0.10 mg /ml 67.04a 60.20a 5.62a 7.40a 0.345a 0.0458a 0.1091a
Least Square Mean 63.08 56.26 4.22 6.20 0.295 0.0411 0.1039

*Values within a column and one type of treatment followed by the same letter are not significantly different at P ≤ 0.05 based on Duncan’s new multiple range test.

378
Z.A. Siddiqui et al. Scientia Horticulturae 249 (2019) 374–382

0.10 mg ml-1 ZnO NPs to plants with P. carotovorum, X. campestris pv.

carotae, M. javanica, A. dauci and F. solani resulted in a greater increase
in chlorophyll, carotenoid, and proline contents than plants sprayed
with 0.05 mg ml-1 ZnO NPs / 0.10 mg ml-1 GO or 0.05 mg ml−1 GO NPs.
The spray of 0.05 mg ml-1 ZnO NPs to plants with P. carotovorum, X.
campestris pv. carotae, M. javanica, A. dauci and F. solani resulted in
statistically similar chlorophyll, carotenoid, and proline contents as
caused by 0.10 mg ml-1 GO. Spray with 0.05 mg ml-1 GO NPs to plants
with test pathogens was least effective in increasing chlorophyll, car-
otenoid and proline contents (Table 3).

3.2.2. Effect on galling and nematode population

Fig. 1. Disease symptoms on foliar parts and roots of carrot.
C = Control; M= Meloidogyne javanica; X= Xanthomonas campestris pv. carotae;
P= Pectobacterium carotovorum; A=Alternaria dauci; F=Fusarium solani
plants without pathogens caused significant increases in chlorophyll,
carotenoid, and proline contents (Table 3). Similarly, application of
0.10 mg ml-1 GO / ZnO NPs (both concentrations) to plants with P.
carotovorum, X. campestris pv. carotae, M. javanica, A. dauci and F. solani
caused a significant increase in chlorophyll, carotenoid and proline
contents over plants inoculated with test pathogens. The spray of
Foliar spray of GO / ZnO NPs at both concentrations caused a sig-
nificant reduction in galling and population of M. javanica (Fig. 2). The
spray of 0.10 mg ml-1 ZnO NPs to plants with M. javanica caused a
higher reduction in galling and nematode population followed by
0.05 mg ml-1 ZnO NPs, 0.10 mg ml-1 GO NPs and 0.05 mg ml-1 GO NPs
(Fig. 2).
3.2.3. Disease indices
Soft rot, leaf blight, leaf spot and root rot indices caused by P.
carotovorum, X. campestris pv. carotae, A. dauci and F. solani respectively
were recorded as 4 (Table 3). Disease indices were reduced to 2 when
0.05 mg ml-1 GO NPs or 0.10 mg ml−1 GO NPs or 0.05 mg ml-1 ZnO
were sprayed on plants with the test pathogens. Disease indices were
reduced to 1 when 0.10 mg ml−1 ZnO was sprayed on plants inoculated
with test pathogens (Table 3).

Table 3
Effect of graphene oxide and zinc oxide nanoparticles on the growth, chlorophyll, carotenoid and proline contents and disease index of carrot.
Treatments Plant length Plant fresh Dry weight(g) Total Chlorophyll Carotenoids (mg / g Proline content Soft rot/ blight / leaf
(cm) weight (g) (mg / g FW) FW) (μmol/gFW) spot / root rot index
Shoot Root

Control Control 71.98b 62.94ab 4.48ghij 6.26defg 0.282hijklm 0.0406efghi 0.0762 q –

M. javanica 56.48m 54.22ijk 2.94no 4.82kl 0.248mno 0.0372hijk 0.0950 n –
P. carotovorum 53.36n 46.15n 2.56op 4.46lm 0.236op 0.0348jkl 0.1008klm 4
X.campestris 58.77klm 51.19l 3.22mn 4.91jkl 0.242nop 0.0361ijkl 0.0985mn 4
A.dauci 52.85n 45.88n 2.22pq 4.19m 0.212p 0.0322l 0.1114def 4
F. solani 44.22o 38.55o 1.97q 3.96m 0.226op 0.0338kl 0.1040hijk 4
GO 0.05 mg/ ml Control 73.58ab 63.92a 4.95defg 6.88cd 0.317bcdefgh 0.0442bcdef 0.0792pq –
M. javanica 62.85fghi 57.42fgh 3.67lm 5.76ghi 0.281ijklm 0.0402fghi 0.0996lm –
P. carotovorum 61.49ghij 53.40jk 3.58lm 5.48hij 0.272jklmn 0.0382ghijk 0.1058ghij 2
X.campestris 61.40hij 56.26ghi 3.97jkl 5.66ghi 0.280ijklm 0.0394fghij 0.1032ijkl 2
A.dauci 58.71klm 52.48kl 3.14mn 5.17ijk 0.253lmno 0.0360ijkl 0.1146bcd 2
F. solani 57.39lm 48.36m 2.90no 4.90jkl 0.258klmno 0.0372hijk 0.1122de 2
GO Control 73.17ab 64.27a 5.38bcd 7.72b 0.345bcd 0.0476ab 0.0823p –
0.10 mg / ml M. javanica 64.75def 59.24cdef 4.56fghi 6.56de 0.312defghi 0.0430bcdefg 0.1022jklm –
P. carotovorum 63.64efgh 57.14fgh 4.08ijkl 6.27defg 0.304fghij 0.0412efgh 0.1079fgh 2
X.campestris 64.29def 58.60def 4.67fgh 6.60de 0.309efghi 0.0423cdefg 0.1056ghij 2
A.dauci 60.84ijk 54.48ijk 3.87kl 5.98efgh 0.283hijkl 0.0396fghi 0.1179ab 2
F. solani 59.63jkl 52.63jkl 3.60lm 5.88fgh 0.292ghijk 0.0404fghi 0.1145bcd 2
ZnO 0.05 mg/ ml Control 73.72ab 64.69a 5.57bc 7.80b 0.348bc 0.0474ab 0.0827p –
M. javanica 65.32de 60.36cde 4.65fgh 6.88cd 0.315cdefghi 0.0435bcdef 0.1028jkl –
P. carotovorum 64.23def 57.75fgh 4.37hijk 6.52def 0.302fghij 0.0417defgh 0.1084efgh 2
X.campestris 65.60cde 58.80def 4.83efgh 6.87cd 0.288ghijk 0.0427bcdefg 0.1060ghij 2
A.dauci 61.29hij 54.77ij 4.05ijkl 6.13efg 0.287ghijkl 0.0402fghi 0.1185ab 2
F. solani 60.86ijk 53.27jkl 3.97jkl 5.97efgh 0.295fghij 0.0407efghi 0.1148bcd 2
ZnO 0.10 mg/ ml Control 75.10a 64.96a 6.45a 8.43a 0.388a 0.0495a 0.0867o 0
M. javanica 67.75c 61.10bc 5.76b 7.65b 0.350b 0.0467abc 0.1075fghi 0
P. carotovorum 66.64cd 60.38cde 5.36bcde 7.28bc 0.341bcde 0.0454abcde 0.1134cd 1
X.campestris 66.29cd 60.69cd 5.62bc 7.64b 0.347bc 0.0462abcd 0.1088efg 1
A.dauci 63.84efg 58.42efg 5.40bcd 6.88cd 0.320bcdefg 0.0430bcdefg 0.1208a 1
F. solani 62.63fghi 55.68hi 5.10cdef 6.54de 0.328bcdef 0.0440bcdef 0.1176abc 1
L.S.D. P = 0.05 NPs 0.86 0.79 0.19 0.23 0.012 0.0016 0.0015
Pathogens 0.94 0.87 0.21 0.25 0.013 0.0018 0.0017
Interaction 2.12 1.95 0.48 0.56 0.029 0.0041 0.0038 –
NPs × Pathogen

*
Values within column followed by different letters are significantly different at P = 0. 05 based on Duncan’s new multiple range test.

379
Z.A. Siddiqui et al.
Fig. 2. Effect of GO NPs and ZnO NPs on galling and population of M. javanica.
4. Discussion
Use of ZnO NPs was found beneficial in improving plant growth,
chlorophyll and carotenoid contents in both pathogen inoculated and
uninoculated plants. The increase in vegetative growth of carrot might
be due to fundamental role of Zn in protection and maintenance of
structural stability of cell membranes (Welch et al., 1982) and its use in
protein synthesis, membrane function, cell elongation and tolerance
(Cakmak, 2000). Zinc is also a cofactor of P-mobilizing enzymes
(phosphatases and phytase) and microbial enzyme dehydrogenases
(Nelson et al., 2008). Bulk ZnO also increased these enzyme activities
but almost half of the nanoparticle efficiency and trends of Zn accu-
mulation were similar for nano and bulk ZnO (Raliya et al.,2016).
Moreover, uptake of nutrients (either nano or bulk) by the plant largely
depends upon the surface biophysical state and its interaction with
plant types (Dimkpa et al., 2017). ZnO NPs were adsorbed in leaves
after spray and taken up through natural nano- or micrometer scale leaf
openings and may accumulate in the vacuole (Raliya and Tarafdar,
2013).
The antifungal activity of ZnO NPs was observed at 0.05 mg ml−1
and 0.10 mg ml−1 against A. dauci and F. solani in potato dextrose agar
medium over control. These fungi with lower concentration of NPs tend
to grow better on the potato dextrose agar plate than with higher
concentration. In this study, ZnO NPs were found to have strong anti-
fungal activity and inhibited the growth of both fungi tested by inter-
fering cell function and causing deformation in fungal hyphae He et al.
(2011). ZnO NPs are known to prevent the conidiophores and conidial
development and deformation of hyphae which ultimately lead to their
death. Application of ZnO NPs in our experiment resulted in reduced
leaf spot and root rot indices, improved growth and chlorophyll con-
tents also confirmed the antifungal effect of ZnO NPs on tested fungi.
ZnO NPs exhibited antimicrobial activity against P. carotovorum and
X. campestris pv. carotae in nutrient agar medium in our study. This can
be explained on the basis of the oxygen species released on the surface
of ZnO, which cause fatal damage to microorganisms (Sunanda et al.,
1998). ZnO NPs react with hydrogen ions to produce molecules of
H2O2. The generated H2O2 can penetrate the cell membrane and kill the
bacteria (Fang et al., 2006). The generation of H2O2 depends strongly
on the surface area of ZnO, which results in more oxygen species on the
surface and the higher antibacterial activity of the smaller nanoparticles
(Yamamoto et al., 2008). Moreover, the antibacterial activity of the
metal oxide nanoparticles mostly appeared on the surface bind with the
thiol (-SH) groups of protein present in the cell wall. The integration of
ZnO NPs with bacteria induce continuous release of membrane lipids
and proteins, which changes the membrane permeability of bacterial
cells and leads to cell lysis (Brayner et al., 2006; Zhang and Chen,
2009). Effect of ZnO NPs on the growth of bacteria was concentration
dependent because better growth, chlorophyll and carotenoides con-
tents were observed in plants sprayed with higher NPs concentration.
380
Scientia Horticulturae 249 (2019) 374–382
Soft rot and leaf blight indices in plants with ZnO NPs were reduced and
resultant increase in plant growth clearly demonstrates the anti-
bacterial nature of ZnO NPs.
Adverse effect of ZnO NPs was also observed on M. incognita eggs
and second stage juveniles. Distributional pattern of ZnO-NPs reveals
that the intestine is the major target tissues for NPs toxicity (Gupta
et al., 2015). Eggs and second stage juveniles were deformed even in
0.05 mg ml-1 concentration within 24 h of incubation and hatching of
second stage juveniles from egg masses was inhibited. Similarly, galling
and nematode multiplication was reduced in plants sprayed with NPs.
Nematodes cuticle is generally rich in sudanophilic lipids (Sood and
Kalra, 1977). The cuticle also consists of weakly acidic mucopoly-
saccharides. Hypodermis and muscles of nematodes contain lipids and
glycogen. In addition, hypodermis also consists of acidic mucopoly-
saccharides (Sood and Kalra, 1977). Toxicity of ZnO NPs is a combi-
nation of dissolved-zinc-caused toxicity and nanoparticle-specific ef-
fects (Sávoly et al., 2016). It is possible the ZnO NPs had adverse effect
on both cuticle and hypodermis of nematodes by affecting lipid, gly-
cogen and mucopolysccharides because disturbed shape of 2nd stage
juveniles of M.javanica was observed under microscope.
In general, the mechanisms of ZnO NPs on living organisms could be
mainly due to physical damage of direct contact, the dissolved zinc ions
and the ROS-mediated mechanism (Hou et al., 2018a). The toxicity of
metal oxide nanoparticles may be related to impairments in DNA
synthesis and repair, as well as to increased production of reactive
oxygen species (Hou et al., 2018b).
Application of GO NPs inhibited the growth of pathogens i.e. P.
carotovorum, X. campestris pv. carotae, A. dauci, F. solani and also had
adverse effect on hatching and mortality of root knot nematode M. ja-
vanica. The addition of GO NPs in culture media not only inhibited the
growth of P. carotovorum, X. campestris pv. carotae, A. dauci, F. solani but
cell walls of bacteria and the mycelia of fungi were disrupted. This
disrupted cell membrane of the fungal spores was verified by examining
the leakage of electrolytes from F. graminearum and F. oxysporum by
Chen et al. (2014). The results indicated that about 57.7% of the total
electrolytes leaked out of the F. graminearum cells and 53.6% out of F.
oxysporum even 300 min after exposure to 500 mg ml_1 GO, suggesting
that GO could indeed disrupt the phospholipids of fungal membranes.
Injury was manifested as an increased leakage of electrolyte. The dis-
ruption of membrane has a large impact on the membrane potential
changes and membrane-associated energy-transducing system, such as
intra- and extra-cellular ATP pools (Lyon and Alvarez, 2008). The ac-
tivation of membrane potential is associated with spore germination.
One of the earliest events in the germination of spores is the activation
of membrane potential (Harris, 2005). Thus, the perturbation of
membrane integrity and the subsequent leakage of electrolytes may
have relationship with inactivation and growth. Moreover,
Sawangphruk et al. (2012) reported that rGO, which is similar to GO in
property, could inhibit the mycelia growth of three fungi, i.e., A. niger,
A. oryzae and F. oxysporum which belong to unicellular eukaryote, one
important member in the microbial family. Antibacterial activity of GO
has also been reported by Liu et al. (2011). Therefore, antimicrobial
activity of graphene, in particular GO, is associated with its unique
optical, electrical, mechanical and thermal properties, such as facile
surface modification, high mechanical strength, good water dis-
persibility, and photoluminescence (Singh et al., 2011).
GO also had adverse effect on the hatching and mortality of M. ja-
vanica. It is possible that toxicity of GO inhibited the hatching of M.
javanica. Hatched 2nd stage juveniles of M. javanica showed mortality
within 24 h of hatching. Observation of dead 2nd stage juveniles under
microscope showed their deformed shape. It is possible that leakage of
electrolytes from body wall of nematodes had resulted in the mortality
of nematodes. Present study reveals that GO inhibited both bacteria and
fungi and reduced root galling and multiplication of M. javanica.
Disease indices of fungal and bacterial pathogens were also reduced
which resulted in increased growth of bacterial or fungal pathogen
Z.A. Siddiqui et al.
inoculated plants. In pot experiment, application of GO also resulted in
the increased growth of pathogen uninoculated plants. Khodakovskaya
et al. (2012) reported the application of carbon nanotubes as regulators
of seed germination and plant growth. Multi walled carbon nanotubes
(MWCNTs) have the ability to enhance the growth of tobacco cell
culture by 55–64% when compared to control at a wide range of con-
centrations from 5 to 500 μg / ml (Khodakovskaya et al., 2012). Carbon
nanotubes (CNTs) can regulate cell division and plant growth by a
unique molecular mechanism that is related to the activation of water
channels (aquaporins) and major gene regulators of cell division and
extension (Khodakovskaya et al., 2012). Therefore, increase in plant
growth to plants without pathogens plus GO can be attributed to above
mentioned reasons.
Proline is a multi-functional amino acid which confers plant defense
against pathogens (Senthil-Kumar and Mysore, 2012). Increase in pro-
line contents was observed in plants inoculated with P. carotovorum, X.
campestris pv. carotae, A. dauci, F. solani and M. javanica. The spray of
ZnO / GO NPs further increased proline, a key osmolyte for maintaining
cellular water content to protect the structure and functions of cellular
organelles (Ahanger and Agarwal, 2017). During stress, proline accu-
mulation is known to maintain osmotic balance, scavenge ROS, stabi-
lize sub-cellular membranes and proteins, and buffer cellular redox
potential (Ashraf and Foolad, 2007; Wahid et al., 2007). Increased
proline content in pathogen-inoculated plants exposed to the spray of
NPs may arise in defense activities. Similarly, an increase in proline
content of Arabidopsis thaliana was observed in response to defense
against pathogens (Fabro et al., 2004; Verslues and Sharma, 2010).
Proline catabolism is known to be enhanced during early stages of plant
defense against invading pathogens (Cecchini et al., 2011); R. solani
infection known to induce proline accumulation in green bean (Ayoubi
and Soleimani, 2014). Faizan et al. (2018) concluded that presence of
ZnO-NPs improved the antioxidant systems and speeded up proline
accumulation that could provide stability to plants and improved
photosynthetic efficiency and may be the possible reasons for improved
growth of pathogen un-inoculated plants.
The above results indicate that the ZnO can display broad spectrum
antimicrobial activity towards tested plant pathogens than GO.
Therefore, the use of ZnO can be expected to serve as a long-term ef-
fective measure for controlling pathogenic fungi, bacteria and plant
parasitic nematodes.
Acknowledgements
Second and third authors are thankful to University Grants
Commission, New Delhi and Aligarh Muslim University Aligarh, India
for the award of University Fellowship to carry out this work.
References
Agrios, G.N., 2005. Plant Pathology, 5th ed. Academic Press, San Diego ISBN:
9780120445653.
Ahanger, M.A., Agarwal, R.M., 2017. Potassium improves antioxidant metabolism and
alleviates growth inhibition under water and osmotic stress in wheat (Triticum aes-
tivum L). Protoplasma. https://doi.org/10.1007/s00709-016-1037-0.
Alghuthaymi, M.A., Almoammar, H., Rai, M., Said-Galiev, E., Abd-Elsalam, K.A., 2015.
Myconanoparticles: synthesis and their role in phytopathogens management.
Biotechnol. Biotechnol. Equip. 29 (2), 221–236 4.
Al-Harbi, A.R., Alsadon, A.A., Khalil, S.O., 1997. Influence of planting date upon growth
and objective component of two carrot cultivars grown in Riyadh region of Saudi
Arabia. J. King Saud. Univ. 9, 257–266.
Ashraf, M., Foolad, M.A., 2007. Improving plant abiotic-stress resistance by exogenous
application of osmoprotectants glycine betaine and proline. Environ. Exp. Bot 59,
206–216.
Ayers, S.H., Rupp, P., Johnson, W.T., 1919. A study of the alkali forming bacteria. Milk
USDA Bull. 782.
Ayoubi, N., Soleimani, M.J., 2014. Possible effects of pathogen inoculation and salicylic
acid pre-treatment on the biochemical changes and proline accumulation in green
bean. Arch. Phytopathol. Plant Prot. 48, 212–222.
381
Scientia Horticulturae 249 (2019) 374–382
Bates, L.S., Waldren, R.T., Teare, I.D., 1973. Rapid determination of free proline for water
stress studies. Plant Soil 39, 205–207.
Bitounis, D., Ali-Boucetta, H., Hong, B.H., Min, D.H., Kostarelos, K., 2013. Prospects and
challenges of graphene in bio-medical applications. Adv. Mater 25, 2258–2268.
Brand, D., Soccol, C.R., Sabu, A., Roussos, S., 2010. Production of fungal biological
control agents through solid state fermentation study in Paecilomyces lilacinus against
root-knot nematodes. Micología Aplicada Int. 22, 31–48 Colegio de Postgraduados
Puebla, México.
Brayner, R., Ferrari-Iliou, R., Brivois, N., Djediat, S., Benedetti, M.F., Fiévet, F., 2006.
Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO na-
noparticles colloidal medium. Nano Lett. 6, 866–870.
Cakmak, I., 2000. Possible roles of zinc in protecting plant cells from damage by reactive
oxygen species. New Phytol. 146, 185–205.
Cecchini, N.M., Monteoliva, M.I., Alvarez, M.E., 2011. Proline dehydrogenase contributes
to pathogen defense in Arabidopsis. Plant Physiol. 155, 1947–1959.
Chen, J., Peng, H., Wang, X., Shao, F., Yuan, Z., Han, H., 2014. Graphene oxide exhibits
broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal
conidia by intertwining and membrane perturbation. Nanoscale 6, 1879–1889.
Dimkpa, C.O., McLean, J.E., Latta, D.E., Manango´n, E., Britt, D.W., Johnson, W.P.,
Boyanov, M.I., Anderson, A.J., 2012. CuO and ZnO nanoparticles: phytotoxicity,
metal speciation and induction of oxidative stress in sand-grown wheat. J. Nanopart.
Res. 14, 1125. https://doi.org/10.1007/s11051-012-1125-9.
Dimkpa, C.O., Bindraban, P.S., Fugice, J., Agyin-Birikorang, S., Singh, U., Hellums, D.,
2017. Composite micronutrient nanoparticles and salts decrease drought stress in
soybean. Agron. Sustain. Dev. 37, 5.
Dye, D.W., 1980. In: Schaad, N.W. (Ed.), Xanthomonas Pages 45-49 in: Laboratory Guide
for Identification of Plant Pathogenic Bacteria. American Phytopathological Society,
St. Paul, MN, pp. 172.
Fabro, G., Kovacs, I., Pavet, V., Szabados, L., Alvarez, M.E., 2004. Proline accumulation
and AtP5CS2 gene activation are induced by plant-pathogen incompatible interac-
tions in Arabidopsis. Mol. Plant Microbe Interact. 17, 343–350.
Faizan, M., Faraz, A., Yusuf, M., Khan, S.T., Hayat, S., 2018. Zinc oxide nanoparticle-
mediated changes in photosynthetic efficiency and antioxidant system of tomato
plants. Photosynthetica 56, 678–686.
Fang, M., Chen, J.H., Xu, X.L., Yang, P.H., Hildebrand, H.F., 2006. Antibacterial activities
of inorganic agents on six bacteria associated with oral infections by two suscept-
ibility tests. Int. J. Antimicrob. Agents 27, 513–517.
Gajjar, P., Pettee, B., Britt, D.W., Huang, W., Johnson, W.P., Anderson, A.J., 2009.
Antimicrobial activities of commercial nanoparticles against an environmental soil
microbe, Pseudomonas putida KT2440. J. Biol. Eng. 3 (9).
Gupta, S., Kushwah, T., Vishwakarma, A., Yadav, S., 2015. Optimization of ZnO-NPs to
investigate their safe application by assessing their effect on soil nematode
Caenorhabditis elegans. Nanoscale Res. Lett. 10, 303.
Harris, S.D., 2005. Morphogenesis in germinating Fusarium graminearum macroconidia.
Mycologia 97 (4), 880–887. https://doi.org/10.3852/mycologia.97.4.880.
He, L., Liu, Y., Mustapha, A., Lin, M., 2011. Antifungal activity of zincoxide nanoparticles
against Botrytis cinerea an d Penicillium expansum. Microbiol. Res. 166, 207–215.
Hou, J., Wu, Y., Li, X., Wei, B., Li, S., Wang, X., 2018a. Toxic effects of different types of
zinc oxide nanoparticles on algae, plants, invertebrates, vertebrates and micro-
organisms. Chemosphere 193, 852–860.
Hou, J., Liu, H., Wang, L., Duan, L., Li, S., Wang, X., 2018b. Molecular toxicity of metal
oxide nanoparticles in danio rerio. Environ. Sci. Technol. 52, 7996–8004.
Kabir, J., Sen, H., Bhattacharya, N., Panda, P.K., Bose, T.K., 2000. Production technology
of vegetable crops. In: ed. In: Bose, T.K., Kabir, J., Das, P., Joy, P.P. (Eds.), Tropical
Horticulture, vol. 2. Naya Prokash, Calcutta, India, pp. 72–240.
Kendrick, J.B., 1934. Bacterial blight of carrot. J. Agric. Res 49, 493–510.
Khodakovskaya, M.V., de Silva, K., Biris, A.S., Dervis hi, E., Villa ga rcia, H., 2012. Carbon
nanotubes induce growth enhancement of tobacco cells. ACS Nano 6, 2128–2135.
Kuan, T.L., Minsavage, G.V., 1985. Detection of Xanthomonas campestris pv. carotae in
carrot seed. Plant Disease 69, 758–760.
Leslie, J.F., Summerell, B.A., 2006. The Fusarium Laboratory Manual, 1st ed. Blackwell
Publishing Ltd, Oxford, London.
Li, F., Jiang, X., Zhao, J., Zhang, S., 2015. Graphene oxide: a promising nanomaterial for
energy and environmental applications. Nano Energy 16, 488–515.
Liu, S.B., Zeng, T.H., Hofmann, M., Burcombe, E., Wei, J., Jiang, R.R., Kong, J., Chen, Y.,
2011. Antibacterial activity of graphite, graphite oxide, graphene oxide, and reduced
graphene oxide: membrane and oxidative stress. ACS Nano 5, 6971–6980.
Lordello, L.G.E., 1984. Nematoideś das plantas cultivadas. Nobel, São Paulo, pp. 314.
Lyon, D.Y., Alvarez, P.J.J., 2008. Fullerene water suspension (nC60) exerts antibacterial
effects via ROS-independent protein oxidation. Environ. Sc.i Technol. 42, 8127–8132.
Mackinney, G., 1941. Absorption of light by chlorophyll solutions. J. Biol. Chem. 140,
315–322.
Nanda, S.S., Papaefthymiou, G.C., Yi, D.K., 2015. Functionalization of graphene oxide
and its biomedical applications. Crit. Rev. Solid State Mater. 40, 291–315.
Nelson, D.L., Lehninger, A.L., Cox, M.M., 2008. Lehninger Principles of Biochemistry.
Macmillan, London.
Nesha, R., Siddiqui, Z.A., 2013. Interactions of Pectobacterium carotovorum pv. car-
otovorum, Xanthomonas campestris pv. carotae and Meloidogyne javanica on the disease
complex of carrot. Intern. J. Veg. Sci. 19 (4), 403–411.
Nunez, J.J., Davis, R.M., 2016. Diseases of Carrot (Daucus Carota L. Subsp. sativus
(Hoffm.).Arcang.). https://www.apsnet.org/publications/commonnames/Pages/
Carrot.aspx.
Ocsoy, I., Paret, M.L., Ocsoy, M.A., Kunwar, S., Chen, T., You, M., Tan, W., 2013.
Z.A. Siddiqui et al.
Nanotechnology in plant disease management: DNA-directed silver nanoparticles on
graphene oxide as an antibacterial against. Xanthomonas perforans. ACS Nano 7,
8972–8980.
osSantos, C.A., Seckler, M.M., Ingle, A.P., Gupta, I., Galdiero, S., Galdiero, M., Gade, A.,
Rai, M., 2014. Silver nano-particles: therapeutical uses, toxicity, and safety issues. J.
Pharm. Sci. 103, 1931–1944.
Peterson, C.E., Simon, P.W., 1986. Carrot breeding. In: Basset, M.J. (Ed.), Breeding
Vegetable Crops. AVI publishing company, Westport (CT), pp. 321–356.
Pfleger, F.L., Harman, G.E., Marx, G.A., 1974. Bacterial blight of carrots: interaction of
temperature, light, and inoculation procedures on disease development of various
carrot cultivars. Phytopathology 64, 746–749.
Prasad, T.N.V.K.V., Sudhakar, P., Sreenivas ulu, Y., Latha, P., Muna swamy, V., Reddy,
K.R., Sreeprasad, T.S., Sajanlal, P.R., Pradeep, T., 2012. Effect of nanoscale zinc oxide
particles on the germination, growth and yield of peanut. J. Plant Nutr. 35, 905–927.
Raliya, R., Tarafdar, J.C., 2013. ZnO nanoparticle biosynthesis and its effect on phos-
phorous-mobilizing enzyme secretion and gum contents in Clusterbean (Cyamopsis
tetragonoloba L.). Agric. Res. 2 (1), 48–57.
Raliya, R., Tarafdar, J.C., Biswas, P., 2016. Enhancing the mobilization of native phos-
phorus in the mung bean rhizosphere using ZnO nanoparticles synthesized by soil
fungi. J. Agricul. Food Chem. 64 (16), 3111–3118.
Sabir, S., Arshad, M., Chaudhari, S.K., 2014. Zinc oxide nanoparticles for revolutionizing
agriculture: synthesis and applications. Sci. World J. 1–8. https://doi.org/10.1155/
2014/925494. Article ID 925494.
Sávoly, Z., Hrács, K., Pemmer, B., Streli, C., Záray, G., Nagy, P.I., 2016. Uptake and
toxicity of nano-ZnO in the plant-feeding nematode, Xiphinema vuittenezi: the role of
dissolved zinc and nanoparticle-specific effects. Environ. Sci. Pollut. Res. 23,
9669–9678.
Sawangphruk, M., Srimuk, P., Chiochan, P., Sangsri, T., Siwayaprahm, P., 2012. Synthesis
and antifungal activity of reduced graphene oxide nanosheets. Carbon 50,
5156–5161.
Schaad, N.W., Jonesand, J.B., Chun, W., 2001. Laboratory Guide for Identification of
Plant Pathogenic Bacteria. 3th. Revista Mexicana de Fıtopatologıa, 343 ed. APS Press,
St. Paul, Minnesota (USA), pp. 373.
Senthil-Kumar, M., Mysore, K.S., 2012. Ornithine-delta-aminotransferase and proline
dehydrogenase genes play a role in non-host disease resistance by regulating pyrro-
line-5-carboxylate metabolism-induced hypersensitive response. Plant Cell Environ.
35, 1329–1343.
Shao, Y., Wang, J., Engelhard, M., Wang, C., Lin, Y., 2010. Facile and controllable elec-
trochemical reduction of graphene oxide and its applications. J. Mater. Chem. 20,
743–748.
Sharma, P.D., 2001. Microbiology. Rastogi and Company, Meerut, India.
382
Scientia Horticulturae 249 (2019) 374–382
Simmons, E.G., 2007. Alternaria: An Identification Manual 6. CBS Biodiversity Series,
Utrercht, the Netherlands, pp. 1–775.
Singh, V., Joung, D., Zhai, L., Das, S., Khondaker, S.I., Seal, S., 2011. Graphene based
materials: past, present and future. Prog. Mater Sci. 56 (8), 1178–1271.
Sood, M.L., Kalra, S., 1977. Histochemical studies on the body wall of nematodes:
Haemonchus contortus (rud., 1803) and Xiphinema insigne Loos, 1949. Zeitschrift für
Parasitenkunde 51, 265–273.
Southey, J.F., 1986. Laboratory Methods for Work with Plant and Soil Nematodes.
Ministry of. Agriculture, Fisheries and Food, pp. 202 Reference Book No. 402.
Stampoulis, D., Sinha, S.K., White, J.C., 2009. Assay-dependent phytotoxicity of nano-
particles to plants. Environ. Sci. Technol. 43, 9473–9479.
Stein, M., Nothnagel, Th., 1995. Some remarks on carrot breeding (Daucus carota sativus
Hoffm.). Plant Breed. 114, 1–11.
Sunanda, K., Kikuchi, Y., Hashimoto, K., Fujishima, A., 1998. Bactericidal and detox-
ification effects of TiO2 thin film photocatalysts. Environ. Sci. Technol. 32, 726–728.
Taylor, A.L., Sasser, J.N., 1978. Biology, Identification and Control of Root-Knot nema-
todes (Meloidogyne Spp.). Depat. Plant Pathol. Raleigh N.C., North, St. Univ. and
USAID, North Carolina State Graphics, pp. 111.
Tulek, S., Dolar, F.S., 2015. Detection and identification of Alternaria species causing
diseases on carrot in Ankara province, Turkey. Scientific Papers. Series B,
Horticulture LIX, 263–268 2015.
Verslues, P.E., Sharma, S., 2010. Proline metabolism and its implications for plant-en-
vironment interaction. Arabidopsis Book 8https://doi.org/10.1199/tab.0140. e0140.
Wahid, A., Gelani, S., Ashraf, M., Foolad, M.R., 2007. Heat tolerance in plants: an
overview. Environ. Exp. Bot. 61, 199–223.
Welch, R.M., Webb, M.J., Loneragan, J.F., 1982. Zinc in membrane function and its role
in phosphorus toxicity. Scaife, A. (Ed.), Proceedings of the Ninth Plant Nutrition
Colloquium 710–715.
Whitehead, N.A., Byers, J.T., Commander, P., Corbett, M.J., Coulthurst, S.J., Everson, L.,
Harris, A.K., Pemberton, C.L., Simpson, N.J., Slater, H., Smith, D.S., Welch, M.,
Williamson, N., Salmond, G.P., 2002. The regulation of virulence in phytopathogenic
Erwinia species: quorum sensing, antibiotics and ecological considerations. Antonie
Van Leeuwenhoek 81, 223–231.
Yamamoto, O., Komatsu, M., Sawai, J., Nakagawa, Z., 2008. Antibacterial activity of ZnO
powder with crystallographic orientation. J. Mater. Sci. Mater. Med. 19, 1407–1412.
Yawalkar, K.S., 1992. Agri-horticultural publishing House, Nagpur, India. Vegetable
Crops of India, 4th ed. pp. 68.
Zhang, H., Chen, G., 2009. Potent antibacterial activities of Ag/TiO2 nanocomposite
powders synthesized by a one-pot sol–gel method. Environ. Sci. Technol. 43,
2905–2910.