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Save Nature to Survive

16(3): 153-165, 2021
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IMPROVEMENT OF GROWTH AND SALINITY TOLERANCE OF

RICE (Oryza sativa L .) CV
L.) CV.. NAVEEN AND L
NAVEEN UNA SANKHI BIOPRIMED
LUNA
WITH OSMOTOLERANT PHYTONIC PLANT GROWTH
PROMOTING BACTERIA

JAYASHREE RATH AND T. K. DANGAR*

Microbiology Laboratory, Crop Production Division,
ICAR-National Rice Research Institute, Cuttack - 753 006, Odisha, INDIA
e-mail:[email protected]

KEYWORDS ABSTRACT
Oryza sp. Five osmotolerant plant growth promoting bacteria (PGPB) viz. Bacillus (SV4, W1), Pseudomonas (NR4),
rice Enterobacter (RP8) and Salinicola (S36) spp. were evaluated for enhancement of growth and osmotolerance of
Naveen rice cv. Naveen (NV) and Luna Sankhi (LS) to select most prospective PGPB to enhance rice production.
Luna Sankhi Biopriming of NV seeds with Salinicola and Pseudomonas spp. increased radical/plumule length, root no.,
seedling fr./dr. wt. (114.29 - 229.32%) more than three (SV4, W1, RP8) other bacteria (84.62 - 140.48%) and
Received on : control (100%). Vegetative and reproductive growth of LS seedlings for NR4 (103.33-181.94%) or S36 (108.19-
30.11.2020 209.39%) and N:1/2P:K fertilizer co-treatments were either comparable or more than those of sole N:1/2P:K
(103.58-195.75%) treatment. LS seedling (7d) growth declined for ≥ 0.21% NaCl (11.61 - 94.74%) and
Accepted on : secondary root emergence for ≥ 0.43% NaCl (0%) in petriplate test but S36 (superior) and NR4 priming reduced
07.06.2021 stress effects, enhanced osmotolerance of seedlings from inherent 6-8 dS/m for 15-21d to 8 dS/m (0.43% NaCl)
for 30d in submerged pot test, 12 dS/m (0.7% NaCl) for 30d by S36 treatment in salinity tank and growth
*Corresponding components by 124.96-400% over control (12dS/m). Investigations identified S36 and NR4 as prospective
author
osmotolerant PGPB for rice improvement under saline and normal conditions.

INTRODUCTION rice genotypes cv. Naveen, Swarna, Khandagiri, IR64 etc. and
other crops (bean, wheat, tomato, chickpea, soybean etc.)
Salinity (osmotic pressure ≥ 0.2 MPa, EC ≥ 4 dS/m, ≥ 40 mM although functionalities differed in intra-/intergenotypic phyto-
NaCl) restricts nutrient availability, growth, development and microbiome (Bal et al., 2012, Sahoo et al., 2014, Pradhan
productivity of rice and plants in general (Shrivastava and and Mishra, 2015, Pahari et al., 2016, Dash and Dangar,
Kumar, 2015, Sampangi-Ramaiah et al., 2020). Rice (Oryza 2019, Egamberdieva et al., 2019, del Carmen Orozco-
sativa L., Poaceae) is most sensitive (threshold 3.0 dS/m) cereal Mosqueda et al., 2020). Furthermore, the salt tolerant plant
to salinity (Kalaiyarasi et al., 2019) which impairs tiller, panicle growth promoting bacteria (PGPB) i.e. Salinicola spp., Bacillus
and spikelet production, floret fertility, grain size, delay heading spp.and Pseudomonas spp. were observed to promote growth
etc. and seedling mortality at 50 mM level (Rad et al., 2012). and salt or metal tolerance of rice (0.3% NaCl, 2% As), wheat
However, rice production needs to be increased for food and other crops (Tiwari et al., 2011, Bal et al., 2012, Pradhan
security in India from 94 to 130 mt and globally from 600 to and Mishra, 2015, Pahari et al., 2016, Zhao et al., 2017,
800 mt by 2025 (Zeigler and Adams, 2008). Thus, Egamberdieva et al., 2019, Mukherjee et al., 2019) through
improvement of rice/crop production in about 6.5 mha (5700 production of different plant growth regulatory substances,
sq. km) inland saline area in India (Maji et al., 2010) and ever- nutrients, antimicrobial substances, exo-polysaccharide (EPS)
increasing global saline land is the primary target to achieve etc. (Pradhan and Mishra, 2015, Shukla et al., 2016). Thus,
the projected food demand. the resident PGPB (especially the phytonic osmotolerant
The ecto- and endophytic plant growth promoting microbes colonizers) of paddy would be best suited for sustainable rice
(PGPM) like Azotobacter, Azospirillum, Bacillus, production/improvement under normal/salt stress conditions
Pseudomonas, Burkholderia, Rhizobium, Pantoea, which is but underutilized to date.
Herbaspirillum, Beauveria, Trichoderma spp. etc. were Importance of PGPB on rice cultivation in saline soils led to
recorded to enhance seed germination, root, shoot, flag leaf evaluate five potent osmotolerant phytonic PGPB (Bacillus
area, tiller, pollination, panicle number, grain wt./number, (SV4, W1), Pseudomonas (NR4), Enterobacter (RP8) and
photosynthesis etc.; salinity (150-200 mM, ~12-15 dS/m Salinicola (S36) spp.) of rice for promotion of growth/production
NaCl), drought, metal, disease etc. resilience; endogenous of high yielding rice cv. Naveen (NV) and Luna Sankhi (LS),
hormone, macronutrient and osmolyte metabolism in different and salinity tolerance of LS to select most promising PGPB for

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futuristic exploitation to improve/sustain rice cultivation under Physico-chemical characters of soil of rice field, pot and
non-stress and osmotic stress conditions. salinity tank experiments
Physico-chemical characters of the rice field (without FYM),
MATERIALS AND METHODS pot and salinity tank soils were analyzed following standard
procedures (Gupta, 2004) through State Soil Testing
Processing of soil, pot, glass vessel and tray for laboratory Laboratory, Bhubaneswar, Odisha, India.
and net house experiments
Processing of seeds for germination and seedling growth
The soil of rice field was collected up to 30 cm depth and evaluation in petridish in laboratory and trays in net house
mixed thoroughly, dried under sun, added with farm yard
The rice cv. Naveen (NV) and Luna Sankhi (LS) seeds were
manure (FYM) ca. 5 t/ha, powdered, sieved to 200 mesh
suspended in distilled water; sedimented seeds (i.e. healthy
size, sterilized at 121ºC for 30 min for 3d consecutively and
filled grains) were collected, washed thoroughly in tap water
used for the experiments. Portions of rice field and FYM mixed
followed by 3 times in sterile distilled water, surface sterilized
soils were used for soil analysis. The plastic pots, glass vessels
for 5 min each in 70% ethanol followed by 0.2% mercuric
and trays were washed with 1% teepol followed by sterile
chloride solution and washed 5 times with sterilized distilled
water to remove traces of detergent, finally with 50% (w/v)
water.
bleaching powder and dried under sun. Inner wall of dried
pots were washed with 70% formaldehyde, air dried and used The seeds were soaked overnight in sterile distilled water for
for the experiments. initiation of germination, spread in sterile (autoclaved)
petridishes lined with a sterile water soaked filter paper,
For evaluation of the bacteria in the net house under normal
maintained in darkness for 2d for plume emergence followed
condition, the pots (20 cm top dia. x 20 cm h) were filled with
by under two fluorescent tube lights of 2100 Lux light intensity
4 kg sterile soil keeping 5 cm top space free and filled with
for 12h photoperiod at 28 ± 2°C for 15d, periodically
water. After 3d, 5 g soil sample from each pot was collected
moistened the paper with sterile distilled water (Nakbanpote
and the composite soil was used for physico-chemical analysis.
et al., 2014, Ng et al., 2012). For seedling production in net
The glass vessels (25 × 9 cm dia. x h) were filled with sterile
house, soil layered (5 cm) trays were moistened with sterile
soil keeping 5 cm top space free. For seedling production in
water with a hand sprayer, 12h pre-soaked seeds were
net house, the trays (60 x 45 x 10 cm l x w x h) were filled with
broadcasted, covered with a thin layer of moist sterile soil and
5 cm deep layer of sterile soil and seeded the pre-soaked
periodically watered by spraying sterile distilled water avoiding
seeds.
stagnation, grown for 15d under sun and used for evaluation.
Processing of perforated pots for salinity tolerance evaluation
Processing of osmotolerant phytonic plant growth promoting
in the net house
bacteria for the experiments
The experiment was designed (Chattopadhyay et al., 2017,
Out of 59 ecto- and edophytic i.e. rhizospheric, rhizoplanic,
2018) in perforated (1 mm dia. at 2 × 2 cm spacing) bottom
endorhyzic, phyllospheric, phylloplanic, endophyllic and
plastic pots (12 cm top dia. x 12 cm h) internally lined with
endocaulic PGPB of three rice (Oryza spp. L., Poaceae) cultivars
compact nylon mess (to prevent soil loss), 20% (v/v) bottom
i.e. O. sativa cv. Naveen (NV, salt sensitive), Swarna Sub-1 (SS,
filled successively one layer each with 10-15, 6-8 and 2-3 mm
submergence tolerant, salt sensitive), Luna Sankhi (LS,
dia. gravel followed by 1.5 kg sand and soil (containing ca. 5t/
moderate salt tolerant, 6-8 dS/m, 15-21d at seedling stage),
ha FYM) mixture (1:1 by wt.), autoclaved at 121ºC, 1h, 3d
and two wild rices i.e. O. latifolia Desv.(OL, salt tolerant, 17.5
consecutively). The bottom of the pots were dipped in NaCl
dS/m, 26d seedling stage) and O. alta Swallen (OA, salt tolerant,
solution of desired salinity, allowed to percolate into pot soil
17.5 dS/m, 33d at seedling stage), selected 5 most prospective
and saturated to attain the desired EC at the top. A piezometer
osmotolerant PGPB and evaluated for growth promotion of
(finely porous tube) was fitted at one side of the pots to monitor
NV and LS without/with salinity stress in the laboratory, net
pot salinity by handheld EC and pH meters.
house and salinity tanks. The prospective organisms viz. LS
Processing of salinity tank for salinity tolerance evaluation rhizoplanic Salinicola sp. (S36) possessing 12% NaCl and sea
Under the shed of transparent polycarbonate sheet (>80% salt tolerance; PGP functions like 1-aminocyclopropane-1-
light transmission), the concrete salinity tanks of 20 x 2 x 1 m carboxylate deaminase (ACCD), inorganic phosphate (IP,
lbh sizes fitted with PVC (poly vinyl chloride) delivery tubes calcium phosphate), organic phosphate (OP, phytate),
along the bottom of the walls and cross connected with lateral ammonia, IAA and siderophore metabolism under non-stress
perforated tubes (1 mm dia., 2 × 2 cm spacing) covered with condition;IP/OP/IAA/siderophore/ACCD/ammonia
compact nylon sleeves to avoid clogging of the holes were metabolism under 6% NaCl stress; OP/IAA/ammonia/
used for evaluation (Chattopadhyay et al., 2017). The tank siderophore metabolism with CO2 stress and biocidal lipase
bottom was filled (15-20 cm) with acid washed gravels (6-8 activity; LS rhizoplanic Bacillus sp. (SV4) having 14 and 16%
mm dia.) covering up to delivery tubes followed by acid washed NaCl and sea salt tolerance; IP/OP/ammonia/ACCD/
coarse sand (10 cm) and 50 cm loose dry soil, sprinkled with siderophore metabolism under normal condition; siderophore
water and compacted to ~1.25 Mg/m3 average bulk density. (CO2, 40ºC), ACCD/ammonia (6% NaCl) metabolism under
The soil samples were collected from 3 tanks for physico- stress and antimicrobial glycosidase, protease, pectinase, lipase,
chemical analysis. Tank soil was salinized by releasing saline cellulase production; NV rhizoplanic Pseudomonas sp. (NR4)
water of desired EC at 3-4d intervals from an overhead tank, with 10 and 12% NaCl and sea salt tolerance; IP/OP/ammonia/
allowed to saturate to obtain desired EC of top soil and ACCD/siderophore/IAA metabolis /N2 fixation under normal
sprinkled with water time to time to avoid evaporation loss. condition; metabolism of IP at 40ºC, phytate under CO2/40ºC

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stress, IAA under NaCl (6%)/CO2/40°C challenge, ammonia seedlings along with different fertilizer doses in pots in net
with 6% NaCl/CO2 stress and protease/cellulase production; house
SS phyllospheric Bacillus sp. (W1) with 14 and 16% NaCl and Healthy, 15d old tray grown rice (cv. Luna Sankhi, LS) seedlings
sea salt tolerance; IP/ammonia/ACCD/siderophore metabolism were dipped separately in bacterial suspensions (1.2 x 108
/N2 fixation at stress-free situation; siderophore (CO2) and cells/ml) for 6h as per BIS, drained off excess inocula, air dried
ammonia (6% NaCl) metabolism under stress and biocidal for 2h and transplanted 3 seedlings/pot (20 cm top dia. x 10
glycosidase/protease/pectinase/lipase/ cellulase enzymes cm h) each with three replications viz. T1: control without any
producers; and OL rhizoplanic Enterobacter sp. (RP8) having fertilizer and seedlings soaked in sterile water, T2: seedlings
10% NaCl/sea salt endurance; IP/OP/ammonia/ACCD/ initially treated with sterile water and received recommended
siderophore/IAA metabolism at stress free condition; dose of fertilizers (RDF) (120:60:60 mg N:P:K/kg soil), T3:
metabolism of IP (6% NaCl), IAA (6% NaCl, CO 2, 40ºC), seedlings received half P (as the bacteria were P solubilizers)
siderophore (CO2) under stress and biocidal lipase enzyme and recommended doses of other fertilizers (N:1/2P:K
producers were used. The bacteria were grown in nutrient 120:30:60 mg/kg soil), T4: seedlings treated with 1.2 x 108
broth (NB, composing (g/l) peptone 5.0, beef extract 3.0, NaCl cells/ml Salinicola sp. (S36), T5: seedlings treated with 1.2 x
3.0, pH 7.0) at 100 rpm in an environmental shaker for 48h at 10 8 cells/ml Salinicola sp. (S36) and N:1/2P:K fertilizers
28±0.1ºC. The cultures were either used directly or (120:30:60 mg/kg soil), T6: seedlings treated with 1.2 x 108
centrifuged at 10000g for 5 min at 4±0.1ºC, washed 3 times cells/ml Pseudomonas sp. (NR4), and T7: seedlings treated
with sterile distilled water by centrifugation and the bacterial with 1.2 x 108 cells/ml Pseudomonas sp. (NR4) and N:1/2P:K
pellet was suspended in sterile distilled water. If required, the fertilizers (120:30:60 mg/kg soil). The plants were grown in
bacterial populations in NB or washed bacteria suspensions the net house under solar radiation with daily maximum
were adjusted with water to ~108 bacteria/ml (A600 0.5) for photosynthetic photon flux density, air temperature and relative
experimentation. humidity (about 1660 µM (m 2/s), 32.6ºC and 70 to 75%,
Assessment of 5 potent bacteria against rice cv. Naveen for respectively). The pots were watered with autoclaved RO
seed germination and seedling growth in petriplate in (reverse osmosis) water to maintain 3-5 cm of standing water
laboratory up to harvest stage (Dash and Dangar, 2019).
PGP effects of the bacteria were evaluated using pre-soaked Growth parameters viz. plant height (cm/plant) and tiller/hill
seeds of cv. Naveen (NV) by seed germination (%) under normal (no./plant i.e. hill) were recorded at panicle initiation (PI) stage
conditions in petriplates in the laboratory. The seeds were after 50d growth i.e. 30d after transplant (DAT); prior to harvest
inoculated by soaking separately with the five (S36, NR4, SV4, (110d) plant height (cm/plant), panicle length (cm/panicle) and
W1 and RP8) potent bacterial suspensions (108 cfu/ml as per leaf area (sq. cm/leaf) were measured and postharvest
Bureau of Indian Standards (BIS) recommendation) for 6h at observations like shoot fr. wt. (g/plant) and dr. wt. (g/plant);
28 ± 2ºC and air-dried for 2h. Bacterized 10 seeds each (3 root length (cm/plant), fr. wt. (g/plant) and dr. wt. (g/plant); total
replications) were taken in separate petridishes lined with a leaves (no./plant), panicle weight (g/panicle) and 100 grain wt.
sterile moist Whatman no.1 filter paper. The petridishes were (g) were recorded after harvest (110d),and plant height (cm)
incubated at 28 ± 2ºC for 5d but 2d under darkness and 3d was measured from the soil surface up to the tip of the top
under fluorescent light (mentioned elsewhere) with 12h light- most shoot each of 3 sampling hills and averaged. Height (cm)
dark cycle in the laboratory. Number of germinated seeds, at maturity was recorded from the soil surface up to the tip of
and plumule and radical lengths were recorded after 5d. All the top most panicle. Total tillers (no.) in each pot were counted
seedlings were blotted to dryness within the filter papers and and average tillers per plant (hill) were recorded. Length of 10
average fr. wt. (g/seedling) was recorded. The seedlings were panicles (arbitrarily selected) was measured (cm) from the neck
air dried under shed for 12h followed by in an oven at 65 ± node to the tip of the top most grain and averaged. Three
0.2ºC to attain constant wt. and average dr. wt. (g/seedling) plants from each treatment were uprooted by loosening the
was noted (Nakbanpote et al., 2014). soil and without damaging the roots. The roots were washed
Evaluation of 5 potent bacteria for growth of rice cv. Naveen thoroughly, length was measured (cm) and averaged, then the
seedlings grown in glass vessel in the laboratory roots were kept in oven (65 ± 2ºC) to attain a constant dr. wt.
The 5d old Naveen seedlings grown in trays in the net house (g). The leaves were separated, dried and leaf area were
were washed thoroughly with sterile distilled water, roots were measured through Licor-300 leaf area meter and average leaf
separately dipped 6h in the potent bacterial (NR4, W1, SV4, area (sq. cm) was noted. Gain yield (g) was recorded from the
S36 and RP8) suspensions (10 8 cfu/ml as per BIS), excess harvest of each pot, dried to 14% moisture content and
inoculum was drained off and dried under fan for 2h. In each obtained average of 100 seed weight (g).
vessel (25 × 9 cm h × dia.) one bacteria primed seedling was Evaluation of the most promising bacteria S36 and NR4 for
planted with 3 replications. The vessels were watered with seed germination and seedling growth of rice cv. Luna Sankhi
sterile water keeping 5 cm water stand and maintained under grown in petriplate in the laboratory under saline condition
fluorescent light (mentioned elsewhere) with 12h light-dark The pre-soaked LS seeds were bacterized by dipping separately
cycle. After 15d growth in vessel, growth parameters viz. shoot in each bacterial suspension (108 cfu/ml as per BIS) for 6h at
length, root length, fresh and dry wt. of shoot and roots were
28 ± 2ºC. Batches of treated seeds (10 each) were incubated
recorded and analyzed to reveal the effects of the bacteria on
separately in sterile petridishes lined with a filter paper
growth of the NV seedlings (Dash and Dangar, 2019).
containing 10 ml sterile 0.21%, 0.43% and 0.87% (w/v) NaCl
Evaluation of S36 and NR4 for growth of rice cv. Luna Sankhi (4, 8, 16 dS/m EC i.e. Eh) along with sole S36 and NR4

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JAYASHREE RATH AND T. K. DANGAR

challenged, and only pre-soaked seeds in sterile deionized (15d) plants were allowed for salinization of desired EC (8dS/
water. The petriplates were arranged in a complete randomized m) for 2d followed by 12dS/m from the reservoirs into the soil
block design with three replications, maintained 2d in darkness tank. On 30d growth parameters viz. plant height (cm/plant),
and 5d under fluorescent light (mentioned elsewhere). Number root length (cm/plant), green leaves (no./plant), affected (dry)
of germinated seeds was counted after 7d, germination rate shoot (no./plant), shoot area (cm2/shoot), no. of tiller/plant, fr.
(%), radical length (cm/seedling), plumule length (cm/seedling), wt. of shoot (g/plant), dr. wt. of shoot (g/plant), fresh wt. of root
fr. wt. (g/seedling), dr. wt. (g/seedling), lateral root (no./seedling) (g/plant) and dr. wt. of root (g/plant) were recorded
were recorded and analyzed (Nakbanpote et al., 2014). (Chattopadhyay et al., 2018).
Evaluation of S36 (Salinicola sp.) and NR4 (Pseudomonas Effects of S36 ectC gene cloned E. coli DH5α
α on seed
sp.) for growth of Luna Sankhi seedlings in perforated pots germination and seedling growth of cv. Luna Sankhi in
under salt stress condition in the net house petriplates under 0.21% NaCl stress
Pre-soaked LS (seedling tolerance 6-8 dS/m for 15-21d) seeds The ectC gene was successfully cloned in E. coli DH5a; salt
were dipped separately in bacterial suspensions (1.2 x 108 tolerance limit of the donor (S36), recipient (E. coli DH5a) and
cells/ml as per BIS) for 6h, air dried for 2h under fan and the clones was evaluated by growing them with different NaCl
sowed 10 seeds in each perforated bottom pots containing concentration (1-12% considering 12% tolerance of S36) in
moist soil without any fertilizer and watered with sterile RO petridish in the laboratory and three positive clones (no. 3, 5,
water. After 15d of germination, pots (3 replications) were 7) with enhanced osmotolerance than the recipient mother
placed on perforated plastic platforms kept in deep trays bacteria were assessed. Effects of the clones on improvement
containing 4 lit (or as required) sterile distilled water (control) of osmotolerance of Luna Sankhi were evaluated in petridish
submerging up to 5 cm basal part and another set (3 in the laboratory (Dash and Dangar, 2019). Pre-soaked Luna
replications) in 4 lit (or as required) sodium chloride solution Sankhi seeds were bioprimed in S36, E. coli DH5a and 3 (no.
(8 dS/m). Water level of the trays were maintained with sterile 3, 5 and 7) cloned bacterial suspensions (1.12 x 108 cells/ml
deionized distilled water and the pots were maintained under as per BIS) for 6h. In 0.21% NaCl (10 ml) containing filter
solar radiation (mentioned elsewhere) in net house. Growth paper lined petridishes, 10 bacteria-primed seeds were
parameters like tillers (no./plant), root length (cm/plant), leaves incubated along with 0.21% NaCl control (non-primed seed)
(no./plant) leaf area (cm2/leaf measured by leaf area meter), fr. and maintained for 2d in darkness and followed by 5d under
wt. of root (g/plant), dr. wt. root (g/plant), fr. wt. shoot (g/plant), fluorescent light (mentioned elsewhere). Seed germination,
dr. wt. shoot (g/plant) and plant height (cm/plant) were radical length (cm/seedling), shoot length (cm/seedling),
recorded after 30d (15 DAT) growth (Nakbanpote et al., 2014, secondary root (no./seedling), dr. wt. (g/seedling) and fr. wt. (g/
Chattopadhyay et al., 2018). seedling) were recorded and analyzed.
Effect of the most potent bacteria S36 on growth of rice cv.
Luna Sankhi seedlings under enhanced salinity (12 dS/m) in RESULTS AND DISCUSSION
saline tanks
Physico-chemical properties of soils used in the experiments
Pre-soaked LS seeds were bacterized in S36 bacterial
Physical properties i.e. pH and EC (Table 1) of the field, pot
suspensions (1.2 x 108 cells/ml as per BIS) for 6h along with
and salinity tank soils were comparable which suggest that
control (water without bacteria), air dried for 2h and planted
FYM had no effect on them but customarily improved NPK
the bioprimed seeds with 15×10 cm (column × rows) spacing
levels in pot and tank soils compared to field soils (Table 1).
in the salinity test tanks containing moist soil. The established However, as field soil was dry but pot/tank soils were collected
Table 1: Physicochemical properties of experimental field, pot and salinity tank soils
Soil parameters Field soil Pot soil Salinity tank soil
Type and texture Sandy-clayey loam Sandy-clayey loam Sandy-clayey loam
pH 6.03 ± 2.20 6.53 ± 2.20 5.90 ± 0.34
EC (dS/m) 0.46 ± 0.25 0.59 ± 0.34 0.64 ± 0.44
Total organic C (%) 1.56 ± 0.45 1.04 ± 0.43 1.18 ± 0.38
Available N (kg/ha) 314.85 ± 6.98 470 ± 0.02 560.72 ± 0.03
Available P (kg/ha) 3.00 ± 1.03 9.00 ± 2.24 6.00 ± 2.88
Available K (kg/ha) 129.06 ± 2.11 166.90 ± 2.26 138.30 ± 2.26
Results are mean of 3 replications ± SE prior to treatment imposition.

Figure 1A: Germination(5d) of rice cv.Naveen seed treated with potent isolates S36,NR 4,SV4,W1,RP8 in petriplate

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Figure 1B: Growth (5d) of isolates rice cv.Naveen seedings under control and treatment with potent isolates S36,NR4,SV4,W1,RP8

Table 2: Effect of potent bacteria on germination and growth of rice cv. Naveen seed treated in laboratory in petriplate
Growth Bacterial treatment LSD CV (%) P-value
parameter (5d) Control S36 NR4 SV4 W1 RP8 -5%
Seed 30 30 30 30 30 30 0 0 0.0000***
germination (%) (100) (100) (100) (100) (100) (100)
Radical length (cm/seedling) 4.2 7.21 8.58 4.2 4.03 5.9 1.16 5.5 0.0033**
(100) (171.67) (204.29) (100) (95.95) (140.48)
Plumule length (cm/seedling) 3.81 5.4 5.52 4.61 4.12 4.6 0.18 4.6 0.0011***
(100) (141.73) (144.88) (121) (108.14) (120.73)
Secondary root (no./seedling) 3.1 6.4 7.1 4.1 4.1 4.2 0.14 4.5 0.0201*
(100) (206.45) (229.32) (132.26) (132.26) 135.48)
Fresh wt. (g/seedling) 0.056 0.066 0.064 0.058 0.058 0.059 0.001 6.2 0.0001***
(100) (117.86) (114.29) (103.57) (103.57) (105.38)
Dry wt. (g/seedling) 0.013 0.02 0.019 0.012 0.011 0.014 0.001 5.8 0.0001***
(100) (153.85) (146.15) (92.31) (84.62) (107.69)
Total no. of seeds per test was 30. S36 = Halomonas sp., NR4 = Pseudomonas sp., W1 = Bacillus sp., SV4 = Bacillus sp., RP8 = Enterobacter sp. Values are means of three replications each of 15
seedlings. Data recorded after 5d. Data in parentheses are percent values. LSD = least significant difference, CV = coefficient of variance. Level of significance = *p < 0.05, **p < 0.01, ***p <
0.001.

Table 3: Effect of the potent bacteria on growth of rice cv. Naveen seedling grown in glass vessel in the laboratory
Growth parameters Control S36 NR4 W1 SV4 RP8 LSD (5%)CV (%) P-value
Bacterial treatment
SL (cm/plant) 23.16 29.1 27.86 25.93 26.33 27.3 2.05 8.4 0.102
(100) (125.48) (120.29) (111.96) (113.69) (117.88)
RL (cm/plant) 7.63 11.03 10.03 8.43 8.33 9.13 1.67 16.1 0.139
(100) (144.53) (131.45) (110.48) (109.17) (119.66)
Fr. wt. of shoot (g/plant) 0.17 0.3 0.3 0.24 0.21 0.26 0.09 19.9 0.052*
(100) (176.47) (176.47) (141.18) (123.53) (152.94)
Dr. wt. of shoot (g/plant) 0.02 0.09 0.07 0.03 0.05 0.05 0.01 15.1 0.013**
(100) (450) (350) (150) (250) (250)
Fr. wt. of root (g/plant) 0.06 0.12 0.08 0.09 0.08 0.07 0.07 17.1 0.017*
(100) (200) (133.33) (150) (133.33) (116.67)
Dr. wt. of root (g/plant) 0.01 0.03 0.02 0.03 0.02 0.02 0.01 12.9 0.013**
(100) (300) (200) (300) (200) (200)
RL = Root length, SL = Shoot length, Values are means of three replications each of 3 seedlings after 15d growth. S36 = Halomonas sp., NR4 = Pseudomonas sp., W1 = Bacillus sp., SV4 = Bacillus
sp., RP8 = Enterobacter sp. Data in parentheses are percent values. LSD = least significant difference. CV = coefficient of variance. Level of significance = *p < 0.05, **p < 0.01, ***p < 0.001.

after 3d watering more native microbial growth would utilize W1, SV4, RP8) on seed germination and initial seedling growth
more available C than former and reduced C content in wet (5d) of Naveen (NV) in plate culture in the laboratory (Table 2,
soil. However, salinity would inhibit growth of salt sensitive Fig. 1A,B) resulted in 100% seed germination for both
resident microbes, thereby, reduce C utilization which would bacterized and control treatments but the bacteria differentially
be reasoned for nominally higher C levels in saline tank than improved (other than radical length for W1 and seedling dr.
pot soil. Available N/P/K in saline tank would be lesser than wt. for SV4 and W1) overall growth (radical length 95.95-
pot soil as salinity reduces nutrient availability in soil 204.29%, plumule length 108.14-144.88%, root no. 132.26-
(Shrivastava and Kumar, 2015). 229.32%, seedling fr. wt. 103.57-117.86%, seedling dr. wt.
Manifestations of seed germination and seedling growth of 84.62-153.85%) of the seedlings over control seeds. Radical
Naveen bioprimed by the bacteria and grown in petriplate elongation (cm/seedling) was more for Salinicola sp. S36 (7.21),
(5d) and glass vessel (15d) without stress in the laboratory Pseudomonas sp. NR4 (8.58) and Enterobacter sp. RP8 (5.90)
Effects of the 5 salt tolerant P mineralizing PGPB (S36, NR4, treatments over control (4.20) which was comparable to

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Table 4: Effects of treatment of the potent bacteria S36 and NR4 on growth of rice cv. Luna Sankhi seedlings grown along with different
fertilizer doses in pots in net house
Growth parameters Treatment LSD CV P-value
T1 T2 T3 T4 T5 T6 T7 (5%) (%)
Plant height at PI (cm/plant) 51.97 60.69 59.96 55.35 57.83 52.71 60.02 1.14 3.3 0.0000***
(100) (116.78) (115.37) (106.5) (111.28) (101.42) (115.49)
Tillers at PI (no./plant) 4.66 5.66 6 5 6.66 5 6.33 0.72 7.3 0.0005***
(100) (121.46) (128.76) (107.3) (142.92) (107.3) (135.84)
Plant height at harvest (cm/plant) 68 76.66 77.18 72.66 76 65.66 75.23 2.61 4.3 0.0039**
(100) (112.74) (113.4) (106.85) (111.76) (96.59) (110.32)
Fresh wt. of shoot at harvest (g/plant) 14.35 22.73 28.09 18.017 29.93 16 24.31 3.99 12.6 0.0001***
(100) (158.39) (195.75) (125.51) (208.57) (111.5) (169.41)
Dry wt. of shoot at harvest (g/plant) 4.79 8.33 8.71 5.64 10.03 5.34 8.22 1.89 14.6 0.0004***
(100) (173.9) (181.84) (117.75) (209.39) (111.48) (171.61)
Root length at harvest (cm/plant) 32.91 36.82 34.64 33.63 37.13 33.44 34.68 1.16 6 0.0061**
(100) (111.88) (105.25) (102.18) (112.82) (100.69) (105.37)
Fresh wt. of root (g/plant) 17.7 25.54 22.24 18.9 28.17 17.93 24.69 1.82 14.8 0.0106**
(100) (144.29) (125.65) (106.78) (159.15) (101.23) (139.49)
Dry wt. of root at harvest (g/plant) 4.54 8.3 6.05 5.11 7.32 5.24 8.26 1.01 23.8 0.0445*
(100) (183.45) (133.26) (112.56) (161.23) (115.41) (181.94)
Leaves at harvest (no./plant) 19 24.66 25.33 20.66 30 20.66 31.33 2.66 6.1 0.0000***
(100) (129.79) (133.32) (108.74) (157.89) (108.74) (164.89)
Leaf area at harvest (cm2/leaf) 14.8 21.86 20.82 18.91 22.39 15.47 20.88 2.44 7.1 0.0001***
(100) (147.7) (140.67) (127.77) (151.28) (104.53) (141.08)
Panicle length at harvest (cm/panicle) 19.53 20.6 20.23 18.86 21.13 18.86 20.18 1.9 5.4 0.1536
(100) (105.48) (103.58) (96.57) (108.19) (96.57) (103.33)
Panicle wt. at harvest (g/plant) 5.47 8.47 7.78 6.62 9.38 6.24 8.45 1.59 12 0.0018***
(100) (154.45) (142.23) (121.02) (171.48) (114.08) (154.48)
100 grain wt. at harvest (g) 1.62 2.39 2.25 1.77 2.81 1.77 2.44 0.25 6.6 0.0000***
(100) (147.53) (138.89) (109.26) (173.46) (109.26) (105.62)
PI = Panicle initiation stage. T1 = Control, T2 = Recommended fertilizer dose (RFD) of N:P:K, T3 = N:1/2P:K, T4 = Sole S36, T5 = S36 + N:1/2P:K, T6 = Sole NR4, T7 = NR4 + N:1/2P:K. Data in the
parentheses are percent values. LSD = least significant difference. CV = coefficient of variance. Level of significance = *p < 0.05, **p < 0.01, ***p < 0.001.

Table 5: Effects of treatment of the potent bacteria S36 and NR4 on germination and growth of rice cv. Luna Sankhi seeds grown in petriplate
in the laboratory under saline condition
Treatment (7d) Seed germi Radical length Plumule length Fr. wt. Dr. wt. Lateral root
nation (%) (cm/ seedling) (cm/ seedling) (g/seedling) (g/seedling) (no./seedling)
Distilled water 30 (100.00) 5.08 (100) 3.82 (100) 0.069 (100) 0.019 (100) 4.00 (100)
NaCl (0.21%) 30 (100.00) 3.21 (63.19) 3.44 (90.05) 0.064 (92.75) 0.018 (94.74) 1.66 (41.50)
NaCl (0.43%) 24 (80.00) 1.94 (38.19) 2.67 (69.90) 0.05 (72.46) 0.016 (84.21) 0
NaCl (0.87%) 14 (46.66) 0.59 (11.61) 1.19 (31.15) 0.037 (53.62) 0.016 (84.21) 0
S36 30 (100.00) 5.20 (102.36) 4.32 (113.09) 0.094 (136.23) 0.042 (221.05) 9.33 (233.25)
S36 + NaCl (0.21%) 30 (100.00) 3.48 (68.50) 3.66 (95.81) 0.074 (107.25) 0.025 (131.58) 8.00 (200.00)
S36 + NaCl (0.43%) 30 (100.00) 2.03 (39.96) 3.36 (87.99) 0.057 (82.61) 0.019 (100.00) 3.33 (83.25)
S36 + NaCl (0.87%) 26 (86.66) 1.19 (23.43) 1.78 (4.60) 0.041 (59.42) 0.018 (94.74) 0
NR4 30 (100.00) 5.29 (104.13) 4.30 (112.57) 0.091 (131.88) 0.041 (215.89) 8.33 (208.25)
NR4 + NaCl (0.21%) 30 (100.00) 3.41 (67.13) 3.57 (93.46) 0.067 (97.10) 0.021 (110.53) 5.66 (141.50)
NR4 + NaCl (0.43%) 28 (93.33) 1.98 (38.98) 2.78 (72.77) 0.055 (79.71) 0.018 (94.74) 0
NR4 + NaCl (0.87%) 22 (73.33) 0.48 (9.45) 0.75 (19.63) 0.038 (55.07) 0.014 (73.68) 0
LSD 6.87 1.09 0.96 0.02 0.01 2.02
CV (%) 3.45 5.09 13.78 7.45 10.16 21.21
P-value 0.32 0.028* 0.023* 0.002** 0.001** 1.98
Total no. of seeds per test was 30. Values are means of three replications each of 15 seedlings. Data in parentheses are percent values. LSD = least significant difference. CV = coefficient of variance.
Level of significance = *p < 0.05, **p < 0.01, ***p < 0.001

Bacillus spp. (SV4 = 4.20 and W1 = 4.03) treatments. Plumule other 3 isolates (0.011-0.014) which was comparable to
growth (cm/seedling) for Pseudomonas sp. NR4 (5.52), control (0.013). Broadly, effects of S36 and NR4 on NV
Salinicola sp. S36 (5.40), Enterobacter sp. RP8 (4.60) and germination and growth did not differ significantly but NR4
Bacillus sp. SV4 (4.61) and W1 (4.12) was more than control was grossly more effective up to 5d seedling growth.
(3.81). Moreover, Salinicola sp. S36 (n = 6.40), Pseudomonas The 5 bacteria treated Naveen seedlings (15d old) grown in
sp. NR4 (n = 7.10) and SV4/W1/RP8 (n = ~ 4) effected more glass vessels (15 DAT) in the laboratory (Table 3, Fig. 2A,B)
secondary root emergence compared to control (n = 3.10). improved shoot and root lengths (cm/plant) viz. Salinicola sp.
S36 (0.066) and RP8 (0.064) augmented fr. wt. (g/seedling) of S36 (29.1, 11.03), Pseudomonas sp. NR4 (27.86, 10.03),
rice seedlings better than other bacteria (~0.058) which was Bacillus sp. W1 (25.93, 8.43), Bacillus sp. SV4 (26.33, 8.33)
comparable to that of control (0.056). Dry wt. (g/seedling) was and Enterobacter sp. RP8 (27.3, 9.13) more than control
also more for S36 (0.020) and NR4 (0.019) treatments than (23.16, 7.63). Enhancement of fr. (0.21-0.30 g/plant) and dr.

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Table 6: Effects of treatment of the potent bacteria S36 and NR4 on growth of rice cv. Luna Sankhi grown in perforated pots under NaCl (8
dS/m) osmoticum
Growth parameters after Treatment LSD CV (%) p-value
15d shock T1 T2 T3 T4 T5 T6 (5%)
Plant height (cm/plant) 40.03 37.35 44.85 41.34 45.26 42.14 1.64 2.2 0.0000***
(100) (93.33) (112.04) (103.27) (113.07) (105.27)
Root length (cm/plant) 18.05 15.97 20.79 18.35 22.4 19.78 1.34 3.9 0.0000***
(100) (88.48) (115.18) (101.66) (124.1) (109.58)
Leaves (no./plant) 7.33 5.66 9.33 7.66 10.33 8.33 1.08 7.4 0.0001***
(100) (77.22) (127.29) (104.5) (140.92) (113.64)
Leaf area (cm2/leaf) 18.89 17.73 23.21 18.9 22.46 20.36 2.48 6.8 0.0037**
(100) (93.86) (122.87) (100) (118.92) (107.78)
Fresh wt. of root (g/plant) 2.03 1.5 2.68 2.53 3.94 3.45 0.68 14.2 0.0002***
(100) (73.89) (132.02) (124.63) (194.09) (169.95)
Dry wt. of root (g/plant) 0.2 0.18 0.33 0.31 0.44 0.37 0.05 17.1 0.0010***
(100) (90) (165) (155) (220) (185)
Fresh wt. of shoot (g/plant) 2.47 2.1 3.75 3.23 4.94 4.11 0.61 10.2 0.0004***
(100) (85.02) (151.82) (130.77) (200) (166.4)
Dry wt. of shoot (g/plant) 0.52 0.48 0.85 0.74 1.41 0.8 0.02 14.3 0.0000***
(100) (92.31) (163.46) (142.31) (271.15) (153.85)
Tillers (No./plant) 2.66 2.66 4.33 4 4.33 4.33 1.1 19.4 0.0313*
(100) (100) (162.78) (150.37) (162.78) (162.78)
T1= Control (distilled water), T2 = NaCl 8dS/m, T3 = NR4 without stress, T4 = NR4 with NaCl 8dS/m, T5 = S36 without stress, T6 = S36 with NaCl 8dS/m. Data in parentheses are percentage (%)
over control. Values are means of three replications. LSD = least significant difference, CV = coefficient of variance. Level of significance = *p < 0.05, **p < 0.01, ***p < 0.001.

Table 7: Effect of treatment of the potent bacterium S36 on growth of rice cv. Luna Sankhi grown in salinity (12 dS/m) tank
Growth parameters treatment T1 T2 LSD (5%) CV (%) P-value
Plant height after 30d (cm/plant) 33.77 (100) 42.20 (124.96) 4.17 3.2 0.0102**
Root Length (cm/plant) 9.28 (100) 16.61 (178.99) 1.82 4.1 0.0023**
Green leaves (no./plant) 6.66 (100) 9.33 (140.09) 1.41 5.1 0.0120*
Affected (dry) leaf (No./plant) 4.33 (100) 2.66 (61.43) 1.74 23.9 0.2002
Leaf area (cm2/leaf) 13.13 (100) 20.8 (158.42) 6.86 11.7 0.0388*
Tiller (no./plant) 2.33 (100) 4.33 (185.84) 0.44 21.2 0.0727
Fresh wt. of shoot (g/tiller) 1.35 (100) 3.11 (230.37) 0.39 4.5 0.0010***
Dry wt. of shoot (g/tiller) 0.26 (100) 0.71 (273.08) 0.12 7.3 0.0027**
Fresh wt. of root (g/tiller) 0.34 (100) 1.36 (400.00) 0.18 13 0.0054**
Dry wt. of root (g/tiller) 0.17 (100) 0.34 (200.00) 0.04 8.4 0.0082**
T1= Control (12 dS/m NaCl), T2 = S36 (12 dS/m NaCl). Data in parentheses are percentage (%) over control. Values are means of three replications and data calculated per plant. LSD = least significant
difference. CV = coefficient of variance. Level of significance *p < 0.05, **p < 0.01, ***p < 0.001

Figure 2a: Growth (15d) of rice seedings cv.Naveen under control(C) Figure 2B: comparative growth of cv.Naveen seedlings under1.
and treatment with S36,NR4,W1,RP8 and SV4 in glass vessel culture control(C) and S36;2. Control (C) and NR4; 3. Control(C) and
in laboratory RP8;4.Control(c) and W1;5.Control(C) and SV4 and SV4 in glass
vessel culture in laboratory
wt. (0.03-0.09 g/plant) of shoot were significantly more for endo-PGPB (Table 2, Fig. 1A,B) which contradicted more (87-
bacteria treatment over bacteria-free plants (0.17 and 0.02 g/ 93%) rice seed germination by P and IAA metabolizing rice
plant) but impacts on root growth were insignificant. Unlike rhizospheric E. gergoviae and C. agropyri (Ng et al., 2012).
plate culture (5d), seedling growth (15d) was better (but However, enhancement of initial (5d) seedling growth by the
insignificant) for S36 than NR4. PGPB over control; superiority of Salinicola sp. (S36) and
Naveen seed germination was not affected by the 5 ecto-/ Pseudomonas sp. (NR4) than other bacteria or control in

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JAYASHREE RATH AND T. K. DANGAR

Figure3A: Vegetative growth of Luna Sankhi plant control and bacteria treatment along with different fertilizer doses.Each treatment effec
t is compares with control(T1)

Figure3B: Panicle initiation stages of Luna Sankhi plant under control and bacteria treatment along with different fertilizer doses

Table 8: Validation of salt tolerance of S36 ectC clones of E. coli 164.71%, shoot dr. wt. 300%) by P metabolizing Enterobacter
DH5αα on NA containing NaCl sp. were observed which were, however, better than those of
Organism NaCl (%) tolerance the 5 PGPB of the present investigation (Dash and Dangar,
S36 12 2019), However, S36 and NR4 were more effective for shoot
E. coli DH5á 5 and root growth (15d) of NV grown in glass vessel soil in the
E. coli DH5á C3 7 laboratory than E. gergoviae (42.43, 20.32%) and C. agropyri
E. coli DH5á C5 7 (28.47, 17.11%) (Ng et al., 2012). As NR4 was isolated from
E. coli DH5á C7 6.5 NV but S36 from LS, therefore, NR4 may have some co-
CD, P = 0.05 0.09 evolutionary relation and might support better (although
insignificant) plumule/radical growth (5d) of NV initially (Table
2, Fig. 1A,B) but S36 being superior PGPB it would surpass
petridish (Table 2, Fig. 1A,B), as well as, support of S36 and subsequent growth (15d) of the NV seedlings (Table 3, Fig.
NR4 for more growth of the seedlings on 15 DAT (30d growth) 2A,B). Thus, the results imply that the 5 bacteria, especially
in glass vessels (Table 3, Fig. 2A,B) proved that NR4 and S36 S36 and NR4 are promising PGPB for rice cultivation.
were superior PGPB of NV. Plant hormone (IAA, zeatin) and P
metabolism by the bacteria (cf. materials and methods i.e. Improvement of growth of Luna Sankhi seedlings treated
MM) might favour root/shoot growth and root formation of with S36 (Salinicola sp.) and NR4 (Pseudomonas sp.) along
NV as recorded from other wild/cultivar/salt tolerant/sensitive/ with different fertilizer doses in pot culture in the net house
drought tolerant/sensitive rice genotypes or plants in general The 2 most promising osmotolerant P solubilizing PGPB viz.
by different ecto-/endo-PGPB (Ng et al., 2012, Pradhan and S36 and NR4 (cf. materials and methods) combined with
Mishra, 2015, Pahari et al., 2016, Walitang et al., 2017, different fertilizer doses were assessed through growth and
Girsowicz et al., 2019, del Carmen Orozco-Mosqueda et al., production of Luna Sankhi (LS, moderate salt tolerant) in pot
2020). Alike the 5 PGPB of present investigation, relative to culture in the net house (Table 4, Fig. 3A, B). Control plants
control more growth of NV seedlings (5d) in petridish (radical without fertilizer (T1) were most poor performers and sole S36
length 207.69%, root no. 700%, plumule length 270%) and (T4) or NR4 (T6) treatments were better (although insignificant)
for 15d growth in glass vessel (root length 161%, shoot length than control but inferior to the remainder treatments. The
130.6%, root fr. wt. 250%, root dr. wt. 500%, shoot fr. wt. recommended fertilizer doses (RFD) (T2) supported better

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Table 9 : Effects of ectC cloned E. coli DH5α

α on germination and growth of Luna Sankhi seedlings in 0.21% NaCl in petriplates
Growth Bacterial treatment along with 0.21% NaCl LSD CV (%) P-value
parameter (5d) Control S36 E. coli E. coli E. coli E. coli
(0.21% DH5α DH5α DH5α DH5α
NaCl) Clone 3 Clone 5 Clone7
Seed germination (%) 30 (100) 30 (100) 30 (100) 30 (100) 30 (100) 30 (100) 0 0 0.001**
Root length (cm/seedling) 3.19 3.49 3.21 3.22 3.23 3.25 0.21 4.1 0.010***
(100) (109.4) (100.63) (100.94) (101.25) (101.88)
Shoot length (cm/seedling) 3.4 3.64 3.58 3.6 3.88 4.05 0.23 4.4 0.011*
(100) (107.06) (105.29) (105.88) (114.12) (119.12)
Secondary root (no./seedling) 1.63 7.92 1.74 2.09 2 1.9 0.23 6.5 0.020*
(100) (485.9) (106.75) (128.22) (122.7) (116.56)
Fresh wt. (g/seedling) 0.061 0.072 0.06 0.063 0.066 0.065 0.021 5.6 0.003**
(100) (118.03) (98.36) (103.28) (108.2) (106.57)
Dry wt. (g/seedling) 0.016 0.025 0.016 0.017 0.018 0.017 0.003 9 0.002**
(100) (156.25) (100) (107.25) (112.5) (107.25)
Results are means of data of 10 seedlings. Data in parentheses are percentage (%) over control. LSD = least significant difference. CV = coefficient of variance. Level of significance *p < 0.05, **p
< 0.01, ***p < 0.001.

(mostly) growth of LS seedlings than N:1/2P:K (T3) and S36 + 3A,B). Furthermore, S36 being a LS colonizers would have
N:1/2P:K (T5) or NR4 + N:1/2P:K (T7) surpassed growth co-evolutionary implication and would better support LS
(except for plant height for S36; fr./dr. wt. of shoot, grain wt. for growth. Moreover, the results proved that S36 is most
NR4) over N:1/2P:K (T3). Evidently, the results (%) of plant promising PGPB for improvement of rice production.
height 111.28 - 116.78 and tiller no. 121.46 - 142.92 at PI Manifestation of seed germination, seedling growth and
stage (30 DAT); root length 105.25 - 112.82, plant height osmotolerance of Luna Sankhi bioprimed by S36 (Salinicola
110.32 - 134.40, root fr. wt. 125.65 - 159.15, root dr. wt. sp.) and NR4 (Pseudomonas sp.) grown in petriplate under
83.45 - 181.94, shoot fr. wt. 158.39 - 208.57, shoot dr. wt. osmotic stress condition
171.61- 209.39, leaf no. 129.79 - 164.89, leaf area 140.67 -
Impact of S36 and NR4 on seed germination and preliminary
151.28, panicle length 103.58 - 108.19, panicle weight
seedling growth of Luna Sankhi (moderate salt tolerant) under
142.23 - 171.48 and 100 grain wt. 105.62 - 173.46 for T 2, T3,
control (0, distilled water), 0.21, 0.43 and 0.87% (or 4, 8, 16
T5 and T7 were more than respective control (T1) (100%).
dS/m or 35.93, 73.58, 148.87 mM) NaCl stress; sole S36 and
Nevertheless, gross results proved that S36 combination with
NR4; and S36 or NR4 combined with 0.21%, 0.43% and
N:1/2P:K (T5) treatment bettered most of the growth parameters
0.87% NaCl stress conditions were evaluated in petriplates in
(%) viz. root length (112.28), tiller no. (142.92), root fr. wt.
the laboratory (Table 5, Fig. 4, 5A,B). Seed germinated was
(159.15), root dr. wt. (161.23), shoot fr. wt. (208.57), shoot dr.
100% in control and 0.21% NaCl but reduced to 80 and
wt. (209.39), leaf no. (157.89), leaf area (151.28), panicle length
46.66% in 0.43 and 0.87% NaCl stress, respectively.
(108.19), panicle wt. (171.48) and 100 grain wt. (173.46) than
Furthermore, 100% seeds germinated in both sole S36 and
other treatments.
NR4, NR4/S36 + 0.21% NaCl and S36 + 0.43% NaCl
Similar to S36 and NR4, more improvement (%) of shoot treatments but 93.33% seeds germinated in NR4 + 0.43%
(102.65-110.39)/root (106.82-134.09) elongation, root fr. NaCl, whereas, S36 + 0.87% NaCl allowed more (86.66%)
(115.60-133.68)/dr. wt. (114.90-141.14), shoot fr. wt. (109.98- seed germination than NR4 + 0.87% NaCl (73.33%). The
133.91), dr. wt. (114.71-138.04), tillering (80-140), leaf no. trends of the effects of the treatments on radical and plumule
(110-130)/area (102-89-108.99), panicle length (109-136.36)/ length, fr. and dr. wt. of seedlings, and lateral root emergence
wt. (103.33-110.83) and 100 grain wt. (100-101.55) for RFD, were similar to seed germination. In control (100%) perspective,
N:1/2P:K, sole Enterobacter sp., N:1/2P:K + Enterobacter sp.
treatments of NV seedlings in the net house in pot culture
were recorded by Dash and Dangar (2019). However, root
growth results for S36 (T5) and NR4 (T7) were lower than those
observed by Khan et al. (2017) who recorded 5% more rice
root length for Burkholderia sp. BRRh-5 with N:1/2P:K
compared RFD fertilized plants. S36 and NR4 with (N:1/2P:K)
(T5, T7) produced 21.46% and 14.38% more tillers, respectively
than RFD treatment (121.46) (T2) which corroborated 17%
more tillers by Burkholderia sp. BRRh-4 with half fertilizer
treated plants compared to un-bacterized RFD supplemented
plants (Khan et al., 2017). The results implicated that bacteria
with 1/2P:N:K would be better for rice growth than bacteria
alone and can reduce at least about half of the P requirement
for rice cultivation (Table 4, Fig. 3A,B). As S36 and NR4 were
effective phosphate and IAA metabolizers than other bacteria
(cf. materials and methods) they would affect better growth of Figure 4: Growth(7d) of rice cv.Luna Sankhi seedling under
the rice cv. Luna Sankhi in non-saline pot culture (Table 4, Fig. control(C) and S36 and NR4 treatment in petriplate

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JAYASHREE RATH AND T. K. DANGAR

(Razzaque et al., 2009, Xu et al., 2011, Nakbanpote et al.,

2014). Enhancement of growth traits by S36 and NR4 over
control, counter action of osmotic shock by the bacterized LS
seedlings conformed to enhancement (over control) of seed
germination, growth, yield of rice and alleviation of inhibition
of higher salinity (10 dS/m) co-inoculated with osmotolerant
B. megaterium A12ag (Sujunya et al., 2009). More root and
shoot length, lateral root emergence and fr./dr. wt. of seedling
by S36/NR4 under NaCl (0.21, 0.43 and 0.87%) corroborated
to those of rice and bean by salt tolerant PGPB, and ACCD
producing rice PGPR viz. Bacillus, Microbacterium,
Methylophaga, Agromyces and Paenibacillus spp. bacterized
Naveen seeds with 150 mM (~0.8%, ~16 dS/m) NaCl (Bal et
al., 2012) stress.
Promotion of growth and salinity endurance of Luna Sankhi
Figure5A: Luna Sankhi seed germination treated with S36 and NR4
under NaCl(0,0.21,0.43,0.87%) stress condition in petriplate(7d)
seedlings treated with S36 (Salinicola sp.) and NR4
(Pseudomonas sp.) grown in perforated pots under saline
stress (8 dS/m) condition in the net house
Effect of the two promising PGPB NR4 and S36 on growth
(15d after treatment) of Luna Sankhi seedlings (tolerance 6-8
dS/m for 15-21d) was evaluated under moderate stress i.e.
8dS/m NaCl (0.43%) compared to stress relaxed conditions
(Table 6, Fig. 6, 7, 8). All growth parameters (but tiller no.) viz.
plant height (40.0 cm/plant), root length (18.05 cm/plant), no.
of leaves (7.33 cm/tiller), shoot area (18.89 cm2/shoot), root fr.
wt. (2.03 g/tiller), root dr. wt. (0.20 g/tiller), shoot fr. wt. (2.47 g/
tiller), shoot dr. wt. (0.52 g/tiller) and tillers no. (2.66/hill) of
control (T1) were impeded under 8 dS/m salt stress (T2) to
93.33, 88.48, 77.22, 93.86, 73.89, 90.00 85.02, 92.31 and
100% (tillers unaffected), respectively. Relative to control (T1)
and osmotic stress (T2), NR4 treatments (T3, T4) augmented
112.04% (plant height) to 165.00% (root dr. wt.) the growth
Figure 5B: Luna Sankhi germinated seeds treated with S36 and NR4 parameters by (T3) and 100% (leaf area) to 155% (root dr. wt.)
under NaCl (0.21,0.43,0.87%) stress condition in petriplates (T4) but improvement levels for S36 (T5, T6) were 113.07%
(plant height ) to 271.15% (shoot dr. wt.) (T5) and 105.27%
results (%) of radical (63.19-11.61)/plumule (90.05-31.15)
length, fr. wt. (92.75-53.62)/dr. wt. (94.74-84.21) and lateral (plant height ) to 185.00% (root dr. wt.) (T6). The results proved
roots (41.50-0) dropped progressively with increasing NaCl that both bacteria augmented osmotolerance (8 to 12 dS/m) of
concentrations (0.21- 0.87%). Sole S36 and NR4 improved the LS seedlings but S36 superseded NR4 both under stress
all seedling growth parameters over control, co-inoculation of free and stress challenged conditions.
the bacteria with various osmoticum levels bettered all growth Negative effect of 8 dS/m NaCl on growth of LS seedlings (T2)
parameters than respective sole salt concentrations and in comparison to control (T1) is a universal stress induced
treatments of S36 + NaCl stresses had superior positive effect growth inhibition effect on plants in general including rice
compared to equivalent osmoticum with NR4. The results (Nakbanpote et al., 2014, Mukherjee et al., 2019). More root/
proved that S36 and NR4 could counter the stress effects on shoot length, leaf no./area, fr./dr. wt. of root/shoot and tiller
seed germination and seedling growth but the former is a production for NR4 (T3) and S36 (T5) than control (T1) and
superior stress alleviator than the latter. 8dS/m NaCl stress (T2), besides, better effect of S36 (T5, T6) than
Although lower salt levels (0.21% NaCl) did not alter seed NR4 (T3, T4) proved osmoprotection function of both PGPBs
germination of Luna Sankhi but negative effects i.e. 80.00% but superiority of S36. Improvement of salt/metal tolerance
(20% reduction) and 46.66% (<50% reduction) on and growth promotion of paddy by phytonic PGPB e.g.
germination for 0.43% and 0.87% osmoticum indicated that Bacillus, Pseudomonas, Halomonas, Corynebacterium,
0.43% (8 dS/m) salt had moderate stress effect and inhibition Enterobacter, Corynebacterium, Bacillus spp. etc. also
of germination by higher salt concentrations would be due to supported overall growth and salt (up to 200 mM NaCl)/
ion toxicity (Ali et al., 2014). Significant improvement of seed drought/metal endurance of different rice cultivars (Naveen,
germination in NaCl (0.43 and 0.87%) co-inoculated with the IR64, moderate osmotolerant Jarava etc.) through osmolyte,
bacteria S36 followed by NR4 in comparison to corresponding osmozyme, IAA, GA3, zeatin; nutrients (N, P, K, S, Ca, Mg) etc.
sole NaCl stresses suggest that they would alleviate osmotic metabolism (Jha et al., 2011, Ng et al., 2012, Jha and
stress of the rice genotype. Similar results on seed germination Subramanian, 2013, Pradhan et al., 2018, Mukherjee et al.,
and seedling growth i.e. root, shoot and dry matter reduction 2019). As S36 was Luna Sankhi rhizoplanic but NR4 was
due to NaCl stress were recorded in rice, cabbage etc. Naveen rhizoplanic colonizer, so more favour of NR4 for

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Figure 6: Growth of cv.Luna Sankhi after 15d transplantation grown in perforated pot containing saline(0.43%) solution in net house

Naveen and S36 for LS would be expected and S36/NR4 may Sankhi grown under salt stress (4 dS/m) in petridish in the
have evolutionary significance with respective host. laboratory
Nevertheless, superiority of S36 for both NV and LS proved NaCl tolerance of the clones (C3, C5, C7) with ectC genes of
that NR4 might be more host specific than S36. Salinicola sp. S36 on NA plates showed that the clones tolerated
Promotion of growth and salinity endurance of Luna Sankhi more NaCl (6.5-7%) than the recipient E. coli DH5a (5%) but
seedlings treated with S36 (Salinicola sp.) grown under salinity lower than the donor S36 (12%) (Table 8). The result proved
stress (12 dS/m) in salinity tanks functional ectC gene of the mother bacterium could be cloned
Growth and salinity resilience of Luna Sankhi seedlings to develop desired osmotolerant PGPB. However, as the donor
(tolerance 6-8 dS/m for 15-21d) treated with the most promising possessed several other stress resilience traits i.e. osmolytes/
S36 under 12 ds/m NaCl (0.70%) stress in salinity tanks up to osmozymes (cf. materials and methods) it would be better
30d were assessed (Table 7, Fig. 9). In control (saline condition) stress tolerant than the single gene cloned E. coli DH5a.
(T1) growth results of plant height (33.77 cm/plant), root length Germination and growth of Luna Sankhi seeds bioprimed with
(9.28 cm/plant), green leaves (6.66/plant), dry leaf (4.33/tiller), the ectC clones and donor PGPB (S36) were checked after 5d
leaf area (13.13 cm2/leaf), tiller no. (2.33/hill), shoot fr. wt. of germination under 0.21% NaCl stress (Table 9). Seed
(1.35 g/tiller), shoot dr. wt. (0.26 g/tiller), root fr. wt. (0.34 g/ germination was unaffected in all treatments but the seedlings
tiller) and root dr. wt. (0.17 g/tiller) were recorded but S36 challenged with the E. coli clones C3 (3.22 cm), C5 (3.23 cm)
treated seedlings grown under salt stress (T2) corresponding and C7 (3.25 cm) had longer roots compared to control (3.19
results were 124.96, 178.99, 140.09, 61.43 (lesser than cm) and DH5a (3.21 cm) but shorter than S36 (3.49 cm).
control i.e. improved), 158.42, 185.84, 230.37, 273.08, Similarly, shoot length (105.88-119.12%), root no. (116.56-
400.00 and 200.00%, respectively, on 30d growth in 12 dS/ 128.22%), seedling fr. wt. (103.28-108.20%) and seedling dr.
m (0.70%) NaCl osmoticum. wt. (107.25-112.50%) increased over control but lower than
Significant growth improvement of LS seedlings by Salinicola the S36 primed seeds. Furthermore, although insignificant, E.
sp. (S36) proved that the bacterium not only enhanced growth coli DH5a was superior than the control and impact of all
of LS but improved osmotolerance from 6-8 to 12dS/m and clones were not identical.
15-21 to 30d (the experiment could not be continued further).
As rice is the most salinity susceptible (threshold 3.0 dS/m) but
an important global cereal crop (Kalaiyarasi et al., 2019),
enhancement of growth and production at higher salinity
would achieve (at least partially) world food security. Higher
NaCl and sea salt (12%) tolerance and PGP functions (including
plant hormone and ACCD metabolism, retention of more
PGP traits (cf. MM) of S36 would be responsible for all round
development of the rice genotype Luna Sankhi beyond its
natural osmotolerance limit and conformed to significant
increase of growth of rice (IR36, Jarava, Naveen etc.) and other
plants under salt stress by salt tolerant PGPB (Egamberdieva,
2014, Orhan, 2016, Mukherjee et al., 2019, del Carmen
Orozco-Mosqueda et al., 2020). The results also proved that
S36 can be exploited both in saline and non-saline conditions Figure7: Evaluation T4(NR4) Pseudomonas sp: and T6(S36,Salinicola
for rice improvement. sp: on growth of Luna Sankhi in perforated pots under saline stress
condition in the net houses.T1= Control(distilled water ),T2=
Salt tolerance of ectC cloned E. coli DH5α
αclones and their Control (NaCl 8ds/m)T3= Only NR4,T4= NR4+NaCl 8ds/
effect on seed germination and seedling growth of cv. Luna m,T5=Only S36,T6=S36+NaCl 8ds/m

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JAYASHREE RATH AND T. K. DANGAR

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