Concurrent effect of phosphorus, nanoparticles and phosphorus solubilizing bacteria influences root morphology, soil enzymes and nutrients uptake in upland rice ( Oryza sativa L.)

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Concurrent effect of phosphorus, nanoparticles
and phosphorus solubilizing bacteria influences
root morphology, soil enzymes and nutrients
uptake in upland rice (Oryza sativa L.)
Ashpakbeg M. Jamadar, B. N. Aravinda Kumar, M. P. Potdar, K. K. Mirajkar,
Hanamant M. Halli, Gurumurthy S. & Raghavendra Nargund
To cite this article: Ashpakbeg M. Jamadar, B. N. Aravinda Kumar, M. P. Potdar, K. K. Mirajkar,
Hanamant M. Halli, Gurumurthy S. & Raghavendra Nargund (20 Feb 2024): Concurrent
effect of phosphorus, nanoparticles and phosphorus solubilizing bacteria influences root
morphology, soil enzymes and nutrients uptake in upland rice (Oryza sativa L.), Journal of Plant
Nutrition, DOI: 10.1080/01904167.2024.2315998
To link to this article: https://doi.org/10.1080/01904167.2024.2315998
Published online: 20 Feb 2024.
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Concurrent effect of phosphorus, nanoparticles and
phosphorus solubilizing bacteria influences root morphology,
soil enzymes and nutrients uptake in upland rice (Oryza
sativa L.)
Ashpakbeg M. Jamadar
a
, B. N. Aravinda Kumar
a
, M. P. Potdar
a
, K. K. Mirajkar
b
,
Hanamant M. Halli
a,c
, Gurumurthy S.
c
, and Raghavendra Nargund
d
a
Department of Agronomy, College of Agriculture, UAS, Dharwad, India;
b
Department of Biochemistry,
College of Agriculture, UAS, Dharwad, India;
c
ICAR- National Institute of Abiotic Stress Management, Pune,
India;
d
ICAR- Indian Institute of Soybean Research, Indore, MP, India
ABSTRACT
Phosphorus and zinc are important nutrient elements of concern in upland
rice production. To determine the suitable combination of phosphorus,
nanoparticles (NPs), and bio-fertilizer application as an alternative nutrient
management approach in upland rice cultivation, a Polyvinyl Chloride
(PVC) column experiment was conducted under controlled conditions in
2015. The treatments consisted of three phosphorus levels, two foliar
sprays of NPs (ZnO and TiO
2
), and PSB seed treatment. The results indi-
cated that the combined application of P (50 kg ha
−1
), ZnO NPs (500 ppm),
and PSB seed treatment (20 g kg
−1
) improved the root system architecture
traits of rice (total root length: 3.73–6.74%, diameter: 8.57–22.22%, and vol-
ume: 3.40–3.70%) at depths of 0–30 cm and 30–60 cm, compared to higher
PþTiO
2
NPs þno PSB. Consequently, this combination led to an enhance-
ment in photosynthetic leaf area (by 3.3%), tiller numbers (by 7%), and dry
matter production (by 35.5%). The favorable root growth resulting from
the application of higher P and PSB enhanced zinc uptake by rice grains
(by 38.44%) and straw (by 26.99%). Furthermore, the supplemental applica-
tion of PSB improved the activity of soil enzymes such as dehydrogenase
and phosphatase. As a result, maximum phosphorus use efficiency (PUE;
31.5%) was achieved through the combined application of P (50 kg ha
−1
)
ZnO NPsPSB. In conclusion, this study demonstrated the beneficial
effects of an integrated approach involving phosphorus, PSB, and metal
oxides (ZnO NPs) as a fertilizer management strategy to enhance the total
biomass and PUE of upland rice.
Abbreviations: PSB: Phosphate Solubilizing Bacteria; PUE: Phosphorous
Use Efficiency; PVC: Polyvinyl Chloride; SEM: Scanning Electron Microscope;
XRD: X-Ray Diffraction; NPS: Nano Particles; SPAD: Soil Plant Analysis
Development; TDM: Total Dry Matter; DM: Dry Matter; DAS: Days After
Sowing.
ARTICLE HISTORY
Received 25 January 2023
Accepted 25 January 2024
KEYWORDS
Nanoparticles; phosphorus;
root system; soil enzymes;
upland rice
Introduction
Rice (Oryza sativa L.) serves as the staple food for nearly 50% of the world’s population and
occupies approximately 11% of the global agricultural land. The demand for rice is expected to
CONTACT B. N. Aravinda Kumar bnakumar@gmail.com Department of Agronomy, College of Agriculture, UAS,
Dharwad, India; Hanamant M. Halli hmhalli4700@gmail.com
� 2024 Taylor & Francis Group, LLC
JOURNAL OF PLANT NUTRITION
https://doi.org/10.1080/01904167.2024.2315998

increase by an additional 70%, particularly in rice-consuming countries, in the near future
(Amanullah et al. 2020). However, the productivity of upland rice, especially in acid soils like alfi-
sols, faces significant challenges due to phosphorus (P) and zinc (Zn) deficiencies (Rehman et al.
2012). P and Zn deficiencies stand out as major nutritional constraints affecting rice growth
(Ismail et al. 2007; Fageria and Baligar 2003). Unfortunately, the utilization of P and Zn fertilizers
is hindered by their expense and limited availability (Halli, Angadi, and Patil 2016). These fertil-
izers quickly form insoluble complexes in soil, rendering them inaccessible to plants (Fageria and
Baligar 2003). Consequently, a balanced approach to P and Zn fertilizer use emerges as a crucial
strategy to enhance upland rice yield and boost farmers’ income (Amanullah et al. 2020).
In response to these challenges, considerable efforts are being directed toward improving P use
efficiency in upland rice production by integrating fertilizer application and other crop manage-
ment practices. Simultaneously, the field of nanotechnology offers promising solutions for
addressing metal and oxide-related issues, serving as essential plant nutrients. Nanoparticles
(NPs) have been characterized as ’magic bullets’ due to their site-specific and targeted activities
(Moghaddasi et al. 2017; Dangi and Verma 2021; Mitra et al. 2023). Notably, the application of
NPs such as zinc oxide (ZnO) and titanium dioxide (TiO
2
) via leaves, at low concentrations, has
shown potential for enhancing crop performance. This is achieved by stimulating enzyme activity,
improving photosynthesis, promoting nutrient uptake, enhancing stress tolerance through root
proliferation, and ultimately boosting crop yield and quality (Chaudhary and Singh 2020).
Remarkably, the application of nano ZnO at concentrations of 1000 ppm for rice, 500 ppm for
wheat, and 150–300 ppm for maize has been linked to improved growth and yield attributes.
These improvements are attributed to the enhanced nutrient uptake, increased antioxidant
enzyme activity, and stimulated root growth (Watson et al. 2015; Da Costa and Sharma 2016;
Rameshraddy et al. 2018). Similarly, studies by Daghan (2018) and Zahra et al. (2017) indicate
that TiO
2
NPs, applied at concentrations of 5–20 ppm for maize and 750 ppm for rice, positively
influence plant height, leaf area, chlorophyll content, dry matter production, and nutrient accu-
mulation. Further evidence, such as that provided by Zheng et al. (2005), showcases the advanta-
geous effects of TiO
2
on spinach, improving root parameters, physiological responses, and
biochemical processes. Optimal concentration selection for nanoparticles is crucial for realizing
the highest benefits for a targeted agro-economic trait (Raliya et al. 2014). The foliar application
of nanoparticle-based micro-nutrients is gaining traction among farming communities due to its
potential to enhance efficiency while minimizing environmental impact, making it a promising
alternative to conventional chemical fertilizers (Rizwan et al. 2017; Hatti et al. 2020). While the
application of bulk fertilizers like ZnO or ZnSO
4
faces solubility challenges, nanoparticles exhibit
unique properties due to their dimensions falling between one and 100 nm (Tarafdar et al. 2014).
These nanoparticles can modify their physico-chemical properties significantly compared to bulk
materials. Their entrance into the plant system occurs through various pathways, and their effect-
iveness hinges on intricate interactions within the plant (P�
erez-de-Luque 2017). Cuticular pores,
with an estimated diameter of around 2nm, provide a potential entry point, while the stomatal
pathway, with a size exclusion limit above 10nm, is the most likely route for nanoparticle pene-
tration (Eichert et al. 2008; Eichert and Goldbach 2008). Interestingly, foliar application of nano-
particles has demonstrated growth enhancement in clusterbean and pearl millet, especially under
arid conditions, as compared to soil application (Tarafdar et al. 2012). Therefore, consideration
should be given to alternative fertilization strategies to maximize upland rice yield.
In light of these considerations, integrating bacterial cultures, such as phosphate solubilizing
bacteria (PSB), as seed inoculants presents a viable approach. PSB can improve nutrient availabil-
ity, including P and Zn, in the rhizosphere through metabolite release and the promotion of soil
enzymes and microbes (Arora et al. 2016). Bacillus subtilis, for example, can solubilize insoluble
P, increasing P concentration in roots and enhancing rice grain uptake (Trivedi et al. 2007).
Employing PSB for seed treatment, combined with foliar NPs application, holds potential for
2 A. M. JAMADAR ET AL.

enhancing P utilization and root growth in rice. Despite separate studies detailing the effects of P,
NPs, and bacterial seed inoculation on cereal crops, there is a lack of information on their com-
bined impact in upland rice. Given this background, the present study investigates the synergistic
use of inorganic inputs (P & NPs; ZnO or TiO
2
) and organic inputs (PSB culture) to explore
their interactive effects on root morphology, growth determinants, and soil enzymatic changes.
This approach aims to enhance phosphorus use efficiency (PUE) in upland rice.
Materials and methods
Experimental location and weather
The experiment was conducted during 2015 (June–December) at Polyhouse of main agricultural
research station, University of Agricultural Sciences, Dharwad, Karnataka, India which is situated
at 15290N latitude, 74590E longitudes and at an altitude of 689 m above mean sea level.
Representative soil of the field was used for the polyhouse study. The minimum and maximum
temperature in the polyhouse during the crop growth period was recorded 25 C and 41 C,
respectively, to reflect the outside environment. The average temperature during the active tiller-
ing and flowering stage ranged from 22-26C.
Physico-chemical properties of the soil’
A composite soil sample of 2.5 tons was collected from the nearby upland rice field of Mugad,
Dharwad, India (latitude 12980N and longitude 77570E). The soil was then air-dried and
sieved through 2 mm mesh. The initial properties of soil were analyzed following the standard
protocols (Table 1).
Synthesis and characterization of ZnO and TiO
2
nano particles (NPs)
The solutions of nano ZnO and TiO
2
were prepared at the Nanotechnology laboratory of
University of Agricultural Sciences, Dharwad, India by dispersing the commercial-grade nano-
powder (Sigma-Aldrich, USA) in deionized Milli-Q water through ultrasonication (100 W,
40 kHz) for 30min using chitosan as substrate. The nano scale suspensions appear as clear solu-
tions at pH of 6.8–7 were used. Magnetic bars were placed in the suspensions for stirring to avoid
aggregation of the particles. X-ray Diffraction analysis (XRD) and UV-Visible spectroscopy (SP-
UV500VDB, Spectrum Instruments) were used to characterize these NPs (Rameshraddy et al.
2017; Mitra et al. 2023).
Experimental setup
PVC tubes of one-meter length and 15 cm diameter were cut vertically into two pieces; the cut
surfaces were joined by tape to facilitate plant root extraction. The bottom of the columns was
capped and holes were opened in the bottom to drain out excess water. About 18 kg of air-dried,
and sieved (2 mm) soil was added to each column. The experiment was arranged [three rates of
recommended P (P
1
at 50% i.e. 25kg ha
−1
, P
2
at 75% i.e. 37.5kg ha
−1
and P
3
at 100% i.e. 50kg
ha
−1
), two foliar sprays of NPs (ZnO @500 ppm and TiO
2
@50 ppm), and two levels of PSB
(seed treatment at 20 g kg
−1
seed and control)] in a factorial completely randomized design with
12 treatment combinations and absolute control (P
0
NP
0
PSB
0
). All the experimental units were
repeated three times. A total of 117 columns were prepared for destructive sampling at three
growth stages (Figure 1).
JOURNAL OF PLANT NUTRITION 3

Table 1. Initial soil properties.
Texture
Course
sand (%)
Silt
(%)
Clay
(%) pH EC (dSm
–1
)
Organic
carbon (g kg
−1
)
Available N
(kg ha
−1
)
Available
P
2
O
5
(kg ha
−1
)
Available
K
2
O (kg ha
−1
) Zinc (mg kg
−1
)
Sandy loam 65.55 16.60 17.85 6.53 0.74 5.6 265.2 43.92 253.73 1.98
Methodology
followed
International
pipette method
pH meter Conductivity
bridge
Walkey and
Black’s Wet oxidation
method
Alkaline
permanganate
method
Olsen’s method Neutral Normal
NH4OAC extraction
method
DTPA extraction method
Reference Piper (1966) Piper (1966) Jackson (1973) Jackson (1973) Subbiah and Asija
(1956)
Jackson (1973) Jackson (1973) Lindsay and Norvell (1978)
4 A. M. JAMADAR ET AL.

Crop management
The upland rice variety ‘Mugad Siri’ (UAS, Dharwad, India, 580005), a popular medium to late
maturing, medium-sized bold grains with higher yield and cooking quality, and tolerant to blast
disease was selected for the study. The seeds were surface sterilized with 70% alcohol and 2%
sodium hypochlorite and washed with sterile distilled water. The surface-sterilized seeds were
then treated with Pseudomonas striata. This strain is a potential phosphobacteria releases tartaric
acid which effectively solubilizes unavailable phosphate. Seed inoculation or soil application of P.
striata improves seedling establishment, root biomass and P availability to the plants (Gaind
2013). The inoculum (20 g kg
−1
seed) with density of the culture was 10
7
viable cells per gram
was thoroughly mixed with 10% jaggery (molasses) solution on a clean polythene sheet. The
slurry with inoculum and seeds were uniformly mixed, and then was shade dried and used for
sowing (Mahua, Raj, and Debtanu 2010). Four healthy seeds were sown (on 27
th
June, 2015) in
each column and one healthy plant was retained after 10 days of sowing. Each column was fertil-
ized with urea [CO(NH
2
)
2
] and muriate of potash (KCl) to supply N and K
2
O at the rate of
100:50 kg ha
−1
respectively, and single super phosphate [Ca(H
2
PO
4
)
2
] levels were varied as per P
treatments. Nitrogen was applied in three split doses viz. 50% at planting; and 25% each at 30
and 60 days after sowing. Two foliar applications of nano ZnO and TiO
2
were carried out at a
concentration of 500 ppm and 50 ppm at 45 and 80 days after sowing (DAS), respectively.
Watering was done uniformly at 40% depletion of available soil moisture throughout the growth
stage of rice. Watering was stopped ten days before the harvest of the crop. To avoid over-expos-
ure to environmental variations in the polyhouse, the columns of each set of sampling were ran-
domly rotated from time to time. The minimum and maximum temperature increased during the
season reaching 23.4 C and 40.5 C respectively at the late grain filling stage. The maximum tem-
perature exceeded 30 C one week before flag leaf emergence and 4–5 days before anthesis.
However, the temperature remained stable in the range of 22–29 C during most of the crop
cycle.
Measurement of root parameters
The roots in each column were sampled at diffeent depths; 0–30cm, 30–60 cm, and 60–90cm at
different growth stages of rice, and were washed with tap water using a 1% solution of sodium
hexameta phosphate (Van Noordwijk 1993) to allow easy separation of roots. Maintained repre-
sentative extra plants under each treatment for destructive sampling to perform both root as well
as shoot observations. Then root traits were scanned using a computer system (Regent-STD
Figure 1. An overview of experimental set up.
JOURNAL OF PLANT NUTRITION 5

1600 þWinRHIZO
TM
2013, Regent instrument, Canada, Quebec) as Halli et al. (2021) reported
corn roots morphology in response to deficit irriagtion.
Measurement of SPAD chlorophyll content
Chlorophyll meter (Soil Plant Analysis Development, Nunes Instruments, Coimbatore-641018,
Tamil Nadu, India) values at 90 DAS were recorded at the middle lamina of the third fully
expanded leaf as this leaf is highly related to the nitrogen status of rice plants (Clements 1964).
Due care was taken to minimize the sample error by taking readings from three leaves of five
representative plants from each treatment (i.e. 53¼15 observations per treatment).
Harvesting
The plants were harvested at physiological maturity around 120 DAS as indicated by hardening
of grains, and yellowing of stem and leaves followed by gradual cessation. Data about plant
height, tiller number, leaf area, and above-ground dry matter production were recorded at the
time of harvest. The plant samples were then oven-dried at 65 C.
Measurement of P and Zn uptake
P and Zn uptake at harvest was computed using the formula;
P or Zn uptake ðkgha−1Þ ¼ PorZn %
ð Þ above ground dry matter ðkgha−1Þ=100

Soil samples collected at 0–30 cm depth in each column were analyzed for available P.
Measurement of PUE
PUE (%) was computed as apparent phosphate recovery efficiency as described by Fageria,
Wright, and Baligar (2014).
PUE %
ð Þ ¼P uptake in fertilized plot kg ha−1
 −P uptake in control plot kg ha−1
  100
P uptake in fertilized plot kg ha−1
 
Dehydrogenase activity
The enzymatic assay of dehydrogenase involves the colorimetric determination of 2,3,5-triphenyl
farmazone (TPF) formed by reduction of 2,3,5-triphenyl tetrazolium chloride by the soil microor-
ganisms. Ten grams of soil sample from top 30 cm of soil column was used to estimate this
enzyme activity. The reduced product, TPF was extracted by methanol and its concentration was
measured using a Spectrophotometer (Labindia, Analytical, UV 3200, Maharashtra, India 400602)
at 485 nm. The enzyme activity was expressed as mg of TPF produced g
−1
of soil when incubated
for 24hr at 37 C (Casida, Klein, and Santoro 1964). The standard graph of different concentra-
tions of TPF was prepared in methanol to include 0, 5, 10, 20, 30, and 40 mg TPF per ml.
Phosphatase activity
The alkaline phosphatase activity was measured by estimating the concentration of p-nitrophenol
as a hydrolyzed product of the substrate para nitrophenyl phosphate (PNP). The reaction mixture
6 A. M. JAMADAR ET AL.

comprising one gram of soil, 0.2 ml toluene, 4 ml modified universal buffer (pH 7.5) and 1 ml of
p-nitrophenol phosphate solution were mixed and incubated at 37 C for one hour. One ml of
0.5 M Cacl
2
and 4ml of 0.5M NaOH were added, swirled and filtered. The enzyme activity was
expressed as lg of p-nitrophenol phosphate hydrolyzed g
−1
of soil hr
−1
at 37± 8 C (Eivazi and
Tabatabai 1979). The standard graph of different concentrations of the p-nitrophenol solution
was prepared to include 0, 10, 20, 30, 40, and 50lg ml
−1
. The intensity of yellow color was red
in a spectrophotometer at 420 nm against the reagent blank.
Data analysis
All the data were tested for normality using PROC Univariate analysis Before ANOVA. The
experimental data obtained were subjected to statistical analysis by adopting Fisher’s method of
analysis of variance as outlined by Gomez and Gomez (1984). The level of significance used in
the ‘F’ test was at 1% and the critical difference (CD) or least significant difference (LSD) values
were used for mean separation are given in the respective parameters, wherever the ‘F’ test was
significant.
Results and discussion
X-ray diffraction (XRD) and UV-Visible spectrophotometer
A typical XRD pattern of ZnO NPs was found in the range 5–50(Figure 2a). The diffraction
peak at 2h with 32.90, 34.50, 38.40, 47.62, 57.28, 65.78, 76.26and 88.10corresponded to
Figure 2. Characterization of nano-particles; ZnO (2a & b) and TiO
2
(2c & d).
JOURNAL OF PLANT NUTRITION 7

the crystal planes of (100), (002), (101), (102), (110), (103) and (200) respectively. The average
size of ZnO NPs was determined as 50 nm with crystalline structure from the width of dominant
peaks (100) and (101) reflections according to Debye-Scherrer’s equation. The broadening of the
peaks can be attributed to the small particle size of the synthesized ZnO. An absorption peak was
observed in each spectrum at 380.8 nm which is the characteristic band for the pure zinc oxide
indicating the high purity of the synthesized ZnO NPs (Figure 2b). Likewise, peaks confirming
the presence of TiO
2
appeared at 2h value ranging from 25.3, 38.3, 48, 50, 62to 78.2corre-
sponding to the crystal planes of (101), (004), (200), (105), and (204) respectively, indicating the
formation of anatase phase of TiO
2
(Figure 2c). The average size of the particles was about
40 nm. UV-vis spectrum of the synthesized titanium dioxide indicated an absorption peak at
338.7 nm (Figure 2d).
Effects on root growth of rice
Individual effects of phosphorus, NPs and PSB on total root length (TRL) at harvest varied at dif-
ferent soil depths (p<0.01; Table 2). Though root observations were performed at different
growth stages of rice but for better interpretation restricted our discussion to only one stage (at
harvest). The maximum TRL (2373 cm pl
−1
) was observed with 100% of recommended P; RP
(P
3
) over other P levels and the increase was up to 14–48% (p<0.01). Likewise, NPs, ZnO appli-
cation had significantly increased the TRL up to 60 cm soil depth (2057 cm) over TiO
2
at
0–30 cm depth. However, both the NPs differed significantly over control (p<0.01). In this study,
foliar application of ZnO NPs increased TRL to an extent of 4.6% and 4.2% at 0–30 and
30–60 cm depth, respectively over TiO
2
. The results are in accordance with the findings of
Table 2. Total root length of upland paddy at harvesting as influenced by application of phosphorus, nanoparticles (NPs)
and PSB.
Treatment
Total root length (cm) at harvest
0–30 cm 30–60 cm 60–90 cm
NP
1
NP
2
Mean NP
1
NP
2
Mean NP
1
NP
2
Mean
P
1
PSB
1
1895cd 1854cd 1874de 1389c 1249cd 1319c 429.3e 829.5b 629.4c
PSB
0
1322d 1274e 1298e 1141d 1066e 1104d 459.5d 473.3d 466.4e
Mean 1609d 1564d 1586c 1265c 1158c 1211c 444.4f 651.4e 547.9c
P
2
PSB
1
2299ab 2091b 2195bc 1521c 1419cd 1470b 709.1b 540.1 cd 624.6c
PSB
0
2009c 1907cd 1958c 1439cd 1413cd 1426b 566.8 cd 457.8d 512.3d
Mean 2154b 1999c 2076b 1480b 1416b 1448b 637.9c 498.9d 568.4b
P
3
PSB
1
2492a 2374ab 2433a 1689a 1684a 1686a 1187a 1107.6a 1147a
PSB
0
2324a 2299ab 2311b 1626b 1621b 1624a 786.7b 655.7c 721.2b
Mean 2408a 2336ab 2373a 1657a 1652a 1655a 987.1a 881.6b 934.3a
2057a 1966b 1467a 1409b 689.8a 677.3a
Mean PSB
1
2228a 2106a 2167a 1533a 1451b 1492a 775.3b 825.7a 800.5a
PSB
0
1885b 1826b 1856b 1402b 1367c 1384b 604.3c 528.9d 566.6b
Control 1038 507.1 289
Factor S. Em. þ
C.D.
(p¼0.01) S. Em. þ
C.D.
(p¼0.01) S. Em. þ
C.D.
(p¼0.01)
P 27.16 S 15.11 S 41.01 S
NP 22.18 S 12.34 S 33.48 NS
PSB 22.18 S 12.34 S 33.48 S
P x NP 38.42 NS 21.37 NS 57.99 NS
P x PSB 38.42 S 21.37 S 57.99 NS
NP x PSB 31.37 NS 17.45 NS 47.35 NS
P x NP x PSB 54.33 NS 30.23 NS 82.02 NS
Treatment v/s control 54.33 S 29.72 S 79.63 S
P
1
at 50% of recommended P (25 kg ha
−1
), P
2
at 75% (37.5kg ha
−1
) and P
3
at 100% (50kg ha
−1
), NP
1
: ZnO @500ppm and
NP
2
: TiO
2
@50 ppm; PSB
1
: with PSB (20 g kg
−1
seed); PSB
0
: without PSB; S: Significant and NS: Non-significant.
8 A. M. JAMADAR ET AL.

Tarafdar et al. (2014) who reported that an increase of 4.2% root length due to the application of
ZnO in pearl millet cv HHB-67. As presumed PSB treatment resulted in higher TRL at all the
depths and the increase was larger at 60–90cm and was 41% higher than the 0–30 and 30–60cm.
This demonstrated the beneficial role of PSB on rice root growth. Interestingly, interaction effect
of P PSB was significant up to 60cm soil depth. These results are in line with the findings of
Mankad et al. (2017) that seed treatment with Zn NPs (50ppm) resulted in improvement of anti-
oxidant enzyme activities, germination and an increase in root length and dry weight of rice. The
root diameter followed the similar trend as that of root length (Table 3, p<0.01), but it was
found to decrease as the soil depth increased. As a result treatment P
3
increased the root diam-
eter (0.99 mm) at 0–30 cm and (0.30 mm) at 30–60cm depth. The results are in agreement with
the findings of Prasad et al. (2012) that ZnO application at 1000 ppm improved the root volume
and root dry weight of peanut. Similarly, root length per volume (RLv) is a measure of the
spread of root for resource capture at specified depths showed significant variations with treat-
ments (Table 4). The ZnO NPs showed higher RLv (5.59 km m
−3
) compared to TiO
2
(5.34 km
m
−3
), and both treatments were significantly different over control (2.82 km m
−3
). In this line
Tarafdar et al. (2014) demonstrated the positive effect of foliar application of Zn on the root
area of pearl millet (improved by 24.2%). Similarly, PSB seed treatment resulted in higher RLv
to an extent of 16.8% at 0–30 cm and 7.8% at 30–60 cm and 41.3% at 60–90cm soil depths over
control (p<0.01). However, RLv was gradually decreased with depth across the treatments
(Table 4). An interaction effect was found to be significant with P
3
PSB
1
and found to have
maximum RLv at 30 cm (6.61 km m
−3
) and at 60 cm depth (4.58 km m
−3
). Hence role of PSB
was witnessed in P solubilization as a result better root spread was observed at high P applica-
tion. Therefore, PSB and ZnO NPs supplements P supply in promoting the root growth of
upland rice.
Table 3. Root diameter of upland paddy at harvest in response to application of phosphorus, nanoparticles (NPs) and PSB.
Treatment
Root diameter (mm) at harvest
0–30 cm 30–60 cm 60–90 cm
NP
1
NP
2
Mean NP
1
NP
2
Mean NP
1
NP
2
Mean
P
1
PSB
1
0.73 cd 0.65d 0.69 cd 0.26c 0.26c 0.26c 0.11d 0.25a 0.18c
PSB
0
0.63d 0.63d 0.63d 0.25c 0.25c 0.25d 0.25a 0.29a 0.27a
Mean 0.68a 0.64a 0.66c 0.26a 0.25a 0.26c 0.28a 0.27a 0.22a
P
2
PSB
1
0.94b 0.87c 0.90b 0.28b 0.28b 0.28bc 0.18c 0.12d 0.15cd
PSB
0
0.79c 0.76 cd 0.78c 0.27b 0.28b 0.27bc 0.21b 0.23b 0.22b
Mean 0.87a 0.81a 0.84b 0.28a 0.28a 0.28b 0.19a 0.18a 0.18b
P
3
PSB
1
1.05a 1.00a 1.02a 0.36a 0.28b 0.32a 0.27a 0.24ab 0.25a
PSB
0
0.96b 0.95b 0.95b 0.28b 0.28b 0.28b 0.17c 0.09e 0.13d
Mean 1.00a 0.97a 0.99a 0.32a 0.28b 0.30a 0.22b 0.16c 0.19b
0.85a 0.81a 0.29a 0.27a 0.20a 0.20a
Mean PSB
1
0.91a 0.84b 0.87a 0.30a 0.27b 0.29a 0.19b 0.20ab 0.20a
PSB
0
0.79c 0.78c 0.79b 0.27b 0.27b 0.27b 0.21a 0.20ab 0.21a
Control 0.43 0.20 0.15
Factor S. Em. þ
C.D.
(p¼0.01) S. Em. þ
C.D.
(p¼0.01) S. Em. þ
C.D.
(p¼0.01)
P 0.02 S 0.008 S 0.04 NS
NP 0.02 NS 0.006 NS 0.03 NS
PSB 0.02 S 0.006 NS 0.03 NS
P x NP 0.03 NS 0.01 NS 0.06 NS
P x PSB 0.03 NS 0.01 NS 0.06 NS
NP x PSB 0.02 NS 0.01 NS 0.05 NS
P x NP x PSB 0.04 NS 0.01 NS 0.08 NS
Treatment v/s control 0.04 S 0.01 S 0.09 NS
P
1
at 50% of recommended P (25 kg ha
−1
), P
2
at 75% (37.5kg ha
−1
) and P
3
at 100% (50kg ha
−1
), NP
1
: ZnO @500ppm and
NP
2
: TiO
2
@50 ppm; PSB
1
: with PSB (20 g kg
−1
seed) and PSB
0
: without PSB; S: Significant and NS: Non-significant.
JOURNAL OF PLANT NUTRITION 9

Table 4. Root length per volume of upland paddy at harvest due to phosphorus, nanoparticles (NPs) and PSB.
Treatment
Root length per volume (km m
−3
) at harvest
0–30 cm 30–60 cm 60–90 cm
NP
1
NP
2
Mean NP
1
NP
2
Mean NP
1
NP
2
Mean
P
1
PSB
1
5.15de 5.03e 5.09c 3.77c 3.39d 3.58c 1.16e 2.25b 0.17bc
PSB
0
3.59f 3.46f 3.52d 3.10d 2.89e 2.99d 1.24d 1.28d 0.12d
Mean 4.37a 4.25a 4.31c 3.43d 3.14d 3.29c 1.20c 1.77b 0.14b
P
2
PSB
1
6.24bc 5.68c 5.96b 4.13b 3.85c 3.99b 1.92c 1.46d 0.16c
PSB
0
5.45c 5.18d 5.32b 3.91c 3.84c 3.87bc 1.54c 1.24d 0.13d
Mean 5.85a 5.43b 5.64b 4.02b 3.84c 3.93b 1.73b 1.35b 0.15b
P
3
PSB
1
6.77a 6.45b 6.61a 4.59a 4.57a 4.58a 3.22a 3.01a 0.31a
PSB
0
6.31bc 6.24bc 6.28a 4.42b 4.40b 4.41a 2.13b 1.78c 0.19b
Mean 6.54a 6.35a 6.44a 4.50a 4.49a 4.49a 2.68a 2.39ab 0.25a
5.59a 5.34a 3.98a 3.82b 1.87a 1.84a
Mean PSB1 6.05a 5.72b 5.89a 4.16a 3.94b 4.05a 2.10a 2.24a 0.21a
PSB0 5.12bc 4.96c 5.04b 3.81bc 3.71c 3.76b 1.64b 1.43c 0.15b
Control 2.82 1.37 0.87
Factor S. Em. þC.D. (p¼0.01) S. Em. þC.D. (p¼0.01) S. Em. þC.D. (p¼0.01)
P 0.07 S 0.04 S 0.11 S
NP 0.06 S 0.03 S 0.01 NS
PSB 0.06 S 0.03 S 0.01 S
P x NP 0.10 NS 0.05 NS 0.01 NS
P x PSB 0.10 S 0.05 S 0.01 NS
NP x PSB 0.08 NS 0.04 NS 0.01 NS
P x NP x PSB 0.14 NS 0.08 NS 0.02 NS
Treatment v/s control 0.14 S 0.08 S 0.02 S
P
1
at 50% of recommended P (25 kg ha
−1
), P
2
at 75% (37.5kg ha
−1
) and P
3
at 100% (50kg ha
−1
), NP
1
: ZnO @500ppm and
NP
2
: TiO
2
@50 ppm; PSB
1
: with PSB (20 g kg
−1
seed) and PSB
0
: without PSB; S: Significant and NS: Non-significant.
Figure 3. Effects of P levels, NPs, and PSB culture on plant height (A), leaf area (B), tiller number (C), and total dry matter (D)
production of upland rice. Values shown are means of three replications ± SE.
10 A. M. JAMADAR ET AL.

Effects on above-ground growth of rice
Variations in growth parameters such as plant height, tiller number, leaf area, and total dry mat-
ter of upland rice is presented in Figures 3A and D. The highest (p<0.05) plant height
(98.0 cm), leaf area (2210 cm
2
pl
−1
), number of tillers (21.08), and total dry matter production
(47.70 g pl
−1
) were found with combined application of P
3
PSB
1
ZnO or TiO
2
compared to
the control (81.80 cm, 14.00, 1500 cm
2
pl
−1
, and 16.91 g pl
−1
, respectively) (Figures 3A and D).
Concerning the individual effect of NPs, ZnO application recorded a greater plant height
(98.11 cm), leaf area (2065 cm
2
pl
−1
), a number of tillers (18.50), and total dry matter (41.67 g
pl
−1
) over TiO
2
. In the present study tiller number was increased to an extent of 7% with the
application of NPs. Seeds treated with PSB had a considerable effect on plant height (98.00 cm),
tiller number (18.94), leaf area (2080 cm
2
pl
−1
), and dry matter than that without seed treatment.
The increment in total dry matter production was to an extent of 35.5%. This increment may be
ascribed to the positive effects of metabolites and growth hormones in improving the rice growth
attributes in response to nanoparticles and PSB. These results are in trustworthiness with the
findings of Fageria, Baligar, and Jones (2011) that plant height is the most sensitive trait to P
deficiency, and increased significantly with the application of different rates of phosphorus.
Similarly, Ahmed et al. (2008) reported that the tiller number in rice was increased with P appli-
cation. Enhanced above ground growth was possibly attributed to the improved root morpho-
logical traits of rice such as, total root length, diameter and volume. Similarly, Samart, Phakamas,
and Chutipaijit (2015) reported the beneficial effects of ZnO and TiO
2
NPs on growth rate and
total dry matter production of rice cultivar Chainat-1. Inoculation of PSB with P increased the
dry biomass production in pearl millet and wheat by 12.5% (Tarafdar et al. 2014). The sufficiency
level of P concentration in rice tissue usually corresponds to the range of 1.4–2.7 g k g
−1
dry mat-
ter (Counce and Wells 1986). In our study, P concentration in rice ranged from 0.13 to 1.56 g
kg
−1
dry matter (data not presented) indicating that dry matter production declined at lower P
levels. This is in agreement with the findings of Akinrinde and Gaizer (2006), who reported the
plant P concentration below critical limits, resulted in lower total dry matter in tropical rice culti-
vars. However, the better growthof rice was also possibly due to improved physiological indicators
such as, chlorophyll meter readings (Figure 4), and nutrients absorption with combined applica-
tion of P
3
, ZnO, and PSB (p<0.05) at 90 DAS over other treatments. Notably ZnO NPs showed
6% increase in chlorophyll content over TiO
2
NPs and 27.22% over control. This is following the
findings of Tarafdar et al. (2014) who showed that foliar application of zinc nano-fertilizer
improved the growth, leaf protein, and chlorophyll contents of pearl millet. This might be due to
better penetration of Zn particles in the plant tissue in the augmentation of photosynthesis.
Figure 4. Effects of P levels, NPs, and PSB culture on chlorophyll meter readings in upland rice.
JOURNAL OF PLANT NUTRITION 11

Therefore, combined application of P, Zn NPs and PSB could benefit the aboveground growth of
upland rice.
P
1
at 50% of recommended P (25 kg ha
−1
), P
2
at 75% (37.5 kg ha
−1
) and P
3
at 100% (50 kg
ha
−1
), NP
1
: ZnO @500 ppm and NP
2
: TiO
2
@50 ppm; PSB
1
: with PSB (20 g kg
−1
seed) and
PSB
0
: without PSB.
P
1
at 50% of recommended P (25 kg ha
−1
), P
2
at 75% (37.5 kg ha
−1
) and P
3
at 100% (50 kg
ha
−1
), NP
1
: ZnO @500 ppm and NP
2
: TiO
2
@50 ppm; PSB
1
: with PSB (20 g kg
−1
seed) and
PSB
0
: without PSB.
Soil enzymes activity and zinc uptake
The quantum of dehydrogenase and phosphatase enzymes activity in soil differed significantly
due to P, NPs, and PSB application (p<0.05; Figures 5A,B). A higher dehydrogenase activity
(13.03 mg TPF g
−1
soil day
−1
) and phosphatase activity (19.52 mg pnp g
−1
soil hr
−1
) were observed
at 35 DAS for P
3
followed by P
2
. Our results are in corroboration with the findings of Fayez and
Mahmoud (2009) and Dubey and Fulekar (2011) that increased P levels had increased dehydro-
genase and phosphatase activities. Foliar application of both nanoformulations influenced the
enzyme activity to an extent of 21–53% over control. Previously, in pearl millet, ZnO enhanced
these enzymes’ activity by 21–77% (Tarafdar et al. 2014). Seed treatment with PSB significantly
increased the dehydrogenase and phosphatase activity (12.22mg TPF g
−1
soil day
−1
; 19.15 mg pnp
g
−1
soil hr
−1
). This improvement may be ascribed to activity of oxido-reductases such as dehy-
drogenases which are involved in oxidative processes in soils and their activity mainly depends
on the metabolic state of the soil biota; thus, acting as good indicators of the soil microbial
Figure 5. Effects of P levels, NPs, and PSB culture on soil dehydrogenase (A), and phosphatase activity (B), grain zn uptake (C),
and straw uptake (D) in upland rice.
12 A. M. JAMADAR ET AL.

activity Kohler et al. (2007). Similarly, rice grain and straw Zn uptake significantly influenced by
the treatments (Figures 5C,D). Among P levels, P
3
recorded a higher Zn uptake by rice grain
(138.87 g ha
−1
) compared to control (85.48 g ha
−1
). Foliar application of ZnO NPs recorded sig-
nificantly higher Zn uptake (131.26 g ha
−1
) compared to TiO
2
(121.70 g ha
−1
). Seed treatment
with PSB did not affect significantly the Zn uptake of rice grain (Figure 5C). Interaction effect
was also not significant for Zn uptake of rice grain. Whereas, Zn uptake by rice straw was influ-
enced by different P levels, and foliar application of NPs (Figure 5D). Among the P levels, P
3
improved the Zn uptake of rice straw (106.58 g ha
−1
) over control (62.62 g ha
−1
). Similarly, the
foliar application of NPs ZnO recorded significantly higher Zn uptake (96.63 g ha
−1
) over TiO
2
and control. Improved Zn uptake of rice grain and straw was probably associated with the forma-
tion of active and prolific roots resulting in increased absorption of micronutrients (Devi et al.
2011). In addition, better nutrient utilization by healthier and vigorous plants under optimum
nutrients availability resulted in higher dry matter accumulation, which ultimately led to an
increase in uptake of nutrients (Qurban et al. 2011). It was also opined that Zn increases the P
utilization by encouraging P metabolism thus resulted in higher availability of P for plant uptake.
Therefore, foliar application of ZnO and seed treatment with PSB promotes soil enzymes activity
and could supplement the uptake of Zn in upland rice.
P
1
at 50% of recommended P (25 kg ha
−1
), P
2
at 75% (37.5 kg ha
−1
) and P
3
at 100% (50 kg ha
−1
),
NP
1
: ZnO @500 ppm and NP
2
: TiO
2
@50 ppm; PSB
1
: with PSB (20 g kg
−1
seed) and PSB
0
: without PSB.
P uptake and phosphorus use efficiency (PUE)
Higher uptake of P in rice grains was noticed in P
3
(15.57 kg ha
−1
) over no P application (8.03kg
ha
−1
) (p<0.05; Table 5). Foliar application of NPs; ZnO and TiO
2
maximized grain P uptake by
Table 5. Phosphorus uptake and phosphorus use efficiency (PUE) of upland paddy as influenced by phosphorus, nanoparticles
(NPs) and PSB.
Treatments
Phosphorus uptake (kg ha
−1
)
PUE (%)Grain Straw
NP
1
NP
2
Mean NP
1
NP
2
Mean NP
1
NP
2
Mean
P
1
PSB
1
8.12f 7.00f 7.60e 6.87c 6.28c 6.58c 11.88d 5.09f 8.49d
PSB
0
8.05f 8.88f 8.47e 6.13c 4.82d 6.48d 8.70e 6.85ef 7.78d
Mean 8.13d 7.94d 8.03c 6.50 5.55 6.03c 10.29c 5.97c 8.13c
P
2
PSB
1
12.58d 13.37c 12.97 cd 7.81b 7.40b 7.60b 22.29b 23.31b 22.80b
PSB
0
10.98e 11.67d 11.32d 7.07b 6.49c 6.78c 16.07c 16.40c 16.23c
Mean 11.78c 12.52c 12.15b 7.44 6.94 7.19b 19.18b 19.85b 19.52b
P
3
PSB
1
18.62a 14.82c 16.72a 9.19a 8.49a 8.84a 31.52a 22.53b 27.03a
PSB
0
16.04b 12.80d 14.42b 7.48b 7.90b 7.69b 22.98b 17.34c 20.16b
Mean 17.33a 13.81b 15.57a 8.33 8.20 8.26a 27.25a 19.94b 23.59a
12.41a 11.41a 7.42a 6.90b 18.91a 15.25b
Mean PSB
1
13.14a 11.73b 12.43a 7.95a 7.39a 7.67a 21.90a 16.98b 19.44a
PSB
0
11.69b 11.12b 11.40a 6.89a 6.40b 6.65b 15.92b 13.53c 14.72b
Control 6.34 5.66 –
Factor S. Em. ± C.D. (p¼0.01) S. Em. ± C.D. (p¼0.01) S. Em. ± C.D. (p¼0.01)
P 0.296 S 0.155 S 0.11 S
NP 0.241 S 0.127 S 0.09 S
PSB 0.241 S 0.127 S 0.09 S
P x NP 0.418 S 0.219 NS 0.15 S
P x PSB 0.418 S 0.219 NS 0.15 S
NP x PSB 0.342 NS 0.179 NS 0.12 S
P x NP x PSB 0.592 NS 0.310 NS 0.21 S
Treatment v/s control 0.590 S 0.302 S 0.21 S
P
1
at 50% of recommended P (25 kg ha
−1
), P
2
at 75% (37.5kg ha
−1
) and P
3
at 100% (50kg ha
−1
), NP
1
: ZnO @500ppm and
NP
2
: TiO
2
@50 ppm; PSB
1
: with PSB (20 g kg
−1
seed) and PSB
0
: without PSB; S: Significant and NS: Non-significant.
JOURNAL OF PLANT NUTRITION 13

12.41 kg ha
−1
and 11.41 kg ha
−1
respectively. Likewise, PSB seed treatment accounted for 13.14 kg
ha
−1
P uptake. Further, interaction effect of P
3
NPs PSB was found significant (p<0.05),
thus recorded a greater P uptake (18.12kg ha
−1
) than other treatment combinations. Similarly, Zinc
uptake in grains was higher at P
3
over other P levels and the increase was in the range of 6.4–18%.
Foliar applied ZnO NPs enhanced the zinc uptake in grains by 8% thanTiO
2
NPs. A similar effect
was noticed with zinc uptake by rice straw (Table 5). Higher uptake values of P and Zn in this study
are in accordance with the findings of Raliya et al. (2014) who reported a higher ZnO accumulation
in leaves as observed in this study. The PUE is commonly defined as the biomass or grain yield pro-
duced per unit of P absorbed by a crop. In the present investigation, significantly higher PUE was
recorded with P
3
(23.59%) (Table 5). A higher PUE was probably a consequence of reduced intense
root competition and better root proliferation, thereby efficient use of applied P was observed.
Contrary, suboptimal application of P resulted in low PUE in lower P application. Ahmed et al.
(2008) reported that P uptake in root and shoot, and PUE were significantly higher in P application
(150 mg kg
−1
) and medium P application in upland rice. Supplemental foliar application of ZnO and
TiO
2
NPs resulted in 19% and 15% increase in PUE over control. Further, PUE increased signifi-
cantly to an extent of 24% with ZnO NPs and 32% with PSB seed treatment. Interaction of between
P
3
ZnO PSB
1
had resulted in a higher PUE (31.52%) followed by P
3
TiO PSB
1
(22.53%).
The seed treatment with PSB culture has resulted in additive effect in terms of higher PUE. In this
study, the increase in root volume was attributed to more number of root hairs which was possibly
contributed to enhanced P uptake and PUE. Moreover, improved soil enzymes activity like dehydro-
genase and phosphatase might enhance the soil organic carbon and P solubility in the soil solution.
The low mobility of phosphorus in soils regulates the root growth for P uptake (Lynch 2007; Fayez
and Mahmoud 2009). This is the study kind of the first attempt to show the concurrent effect of soil
application of P, foliar supply of NPs, and seed treatment with PSB in improving the P uptake and
PUE of upland rice.
Conclusion
The combined use of phosphorus and nanoparticles in supplementation with PSB has the poten-
tial to improve root system architecture traits and growth attributes of upland rice. The present
study demonstrates that the efficiency of phosphorus use in upland rice can be enhanced by the
soil application of P (50kg ha
−1
), foliar supplementation of nano ZnO (500 ppm), and seed treat-
ment of PSB (20g kg
−1
). There is a marked increase in soil enzymatic activity, as well as root
ramification, leading to maximized root volume and crop growth. This allows the crop to explore
phosphorus and zinc nutrients to improve the development of aboveground growth. Although
high levels of P in the soil are often responsible for Zn deficiencies, but an optimum nutrient bal-
ance could be achieved by using the foliar application of nano ZnO and PSB. Besides saving the
cost of nutrients (P & Zn), this integrated approach could minimize the adverse effects of excess
fertilizers on the environment.
Acknowledgments
The authors express their gratitude to Dr C.D. Lokhande, Emeritus Professor of Department of Physics, Shivaji
University, Kolhapur, India for their help in interpretation in nano sciences. This research was supported by the
University of Agricultural Sciences, Dharwad, Karnataka to conduct field experiments and laboratory analysis.
Authors’ contributions
A.M.J., B.N.A., M.P.P., K.K.M., and H.M.H. planned, designed and conducted the field experiments, lab analysis
and writing of manuscript; G.S and R.N. performed statistical analysis of the data, manuscript writing, editing and
preparation of graphs.
14 A. M. JAMADAR ET AL.

Disclosure statement
There are no conflicts of interest between authors.
Funding
This research was funded by the University of Agricultural Sciences, Dharwad for experimentation and laboratory
analysis. The authors are grateful to the Department of Agronomy and Bio-chemistry, University of
Agricultural Sciences, Dharwad, India for facilitating needful requirements to conduct the experiment.
Data availability statement
All data are available in the manuscript.
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