Physiol Mol Biol Plants (November–December 2019) 25(6):1483–1495
https://doi.org/10.1007/s12298-019-00710-3
RESEARCH ARTICLE
Resource partitioning in the rhizosphere by inoculated Bacillus
spp. towards growth stimulation of wheat and suppression of wild
oat (Avena fatua L.) weed
Anupma Dahiya1 • Ruchi Sharma1 • Swati Sindhu1 • Satyavir S. Sindhu1
Received: 27 May 2019 / Revised: 22 August 2019 / Accepted: 28 August 2019 / Published online: 28 September 2019
Ó Prof. H.S. Srivastava Foundation for Science and Society 2019
Abstract Common wheat (Triticum aestivum L.) is one of
the most important agricultural crop, which provides direct
source of food for humans. Besides abiotic stresses, weeds
pose a significant challenge to successful crop production.
Avena fatua (wild oat) is one of the most common dam-
aging grass weed, which causes 17–62% losses in yield of
winter wheat. Excessive use of herbicides to control wild
oat has resulted in serious environmental and human health
hazards. Therefore, biological control of weeds is required
to cope up with the increasing food demand and to attain
self-sustainability. In this study, eighty eight rhizobacterial
isolates were isolated from rhizosphere soil samples col-
lected from Rewari and Hisar districts. After screening of
the isolates, only thirty isolates showed in vitro antago-
nistic and herbicidal activities. The selected antagonistic
isolates were further tested for production of IAA and
ALA, and utilization of ACC. Bacterial isolates BWA18
and RWA52 produced 53.80 and 19.18 ug ml-1 IAA,
respectively and high ALA production was shown by iso-
lates HCA3 and RCA3. Five isolates i.e., BWA20,
BWA23, BWA29, BWA38 and RCA3 showed significant
ACC utilization. Inoculation of selected bacterial isolates
BWA18, RWA69 and SYB101 showed significant increase
in root dry weight (RDW) and shoot dry weight (SDW) of
wheat plants under pot house conditions, and decreased
RDW and SDW of A. fatua weed as compared to RDF-
amended uninoculated soil at 25 DAS (days after sowing).
Bacterial isolates RWA69 and SYB101 caused significant
increase in RDW and SDW of wheat growth at 50 DAS,
& Satyavir S. Sindhu
sindhuss58@gmail.com; sindhuss@hau.ac.in
1
Department of Microbiology, CCS Haryana Agricultural
University, Hisar, Haryana 125004, India
whereas their inoculation decreased RDW and SDW of A.
fatua. Thus, seed bacterization with bacterial isolates
RWA52, RWA69 and SYB101 caused significant increase
in RDW and SDW of wheat, whereas their inoculation
caused significant decrease in RDW and SDW of A. fatua.
The best performing bacterial isolates RWA52 and
RWA69 were identified as Bacillus siamensis and Bacillus
endophyticus using 16S rRNA analysis. The promising
rhizobacterial isolates could further be tested for the bio-
herbicidal activity and plant growth promotion effects
under field conditions before their use as bioherbicides.
Keywords Wheat  Avena fatua  Resource partitioning 
Rhizosphere bacteria  Bacillus sp.  Bioherbicides
Introduction
More than 50% of the world energy intake is provided by
cereal crops such as wheat, rice and maize. Among these
crops, wheat (Triticum aestivum L.) is the most important
source of staple food for ever-increasing human population
in the world, which occupies more than 41% of crop area
worldwide (Gooding and Davis 1997; Curtis 2002). The
limited nutrient resources essential for crop growth are
depleted by the weeds, resulting in significant loss in the
biomass and yield of different crops. Moreover, weed’s
interference also increases the cost of production along
with reduction in the quality of produce. Type of weed
species, weed density and the time of emergence of weeds
affect the competition of crop with the weeds for the
resources i.e., space, light, moisture and nutrients under
field conditions.
Avena fatua L. (wild oat) is one of the most common
and economically damaging herbicide-resistant grasssy
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weed that infests a large number of crops around the world
(Sharama and Vanden 2008). This weed is widely dis-
tributed in diverse agro-ecosystems (Holm et al. 1977) and
represents a serious economic threat to crop yields due to
their unique seed traits, high competitiveness, staggered
germination, allelopathic potential and ability to persist in
the soil seed bank. Weed infestation in wheat causes about
21% yield loss depending on crop species and weed den-
sity. Herbicides such as isoproturon, clodinafop-propargyl,
fenoxaprop, pinoxaden, accord plus (fenoxaprop ?
metribuzin), sulfosulfuron and atlantis (meso ? iodosul-
furon) are applied for control of wild oat, but resistance to
several commonly used herbicides is becoming more
prevalent recently. Moreover, excessive spraying of her-
bicides and agrochemicals for weed control is environ-
mentally unsafe and uneconomical, and has resulted into
considerable pollution of soil, water and air. Therefore, it is
necessary to develop cost effective, eco-friendly and sus-
tainable weed management biocontrol practices in which
microorganisms or their products could be used for parti-
tioning of bioresources (nutrients, light, water and space) to
promote the growth of crops and simultaneously to sup-
press the growth of weed species (Weller 1988; Lakshmi
et al. 2015; Sindhu et al. 2018; Zorner et al. 2018).
Several rhizosphere bacteria have been identified which
possess the bioherbicidal activity and these genera include
species of Acinetobacter, Achromobacter, Alcaligenes,
Azospirillum, Bacillus, Burkholderia, Enterobacter, Pseu-
domonas, Ralstonia, Serratia and Rhizobium etc. (Kennedy
et al. 1991; Ahemad and Kibret 2014; Sindhu et al. 2018;
Phour and Sindhu 2019). Some of these rhizosphere bac-
teria were found to suppress the growth of weed species by
producing phytohormones such as indole acetic acid (IAA)
and d-aminolevulinic acid (ALA), which affect different
plant developmental processes in plants. The high con-
centration of phytohormones has been reported to
adversely affect the germination and growth of weeds
(Barazani and Friedman 1999; Mohan Babu et al. 2003;
Sindhu and Sehrawat 2017). ALA produced by rhizobac-
teria has also been reported as an effective natural
biodegradable herbicide (Sasikala et al. 1994) along with
its growth-promoting effect on different crops and veg-
etables (Sasaki et al. 1993; Phour et al. 2018). In addition,
rhizosphere bacteria exhibiting ACC (1-aminocyclo-
propane-1-carboxylate) deaminase activity have been
found quite beneficial under abiotic and biotic stress con-
ditions, leading to promotion of plant growth by reducing
production of stress-induced ethylene (Glick 2005;
Chaudhary and Sindhu 2017; Saikia et al. 2018).
These biocontrol strategies are highly compatible with
the sustainable agriculture and for conservation of natural
resources. In earlier studies, rhizobacterial isolates were
obtained that showed suppressive properties against green
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Physiol Mol Biol Plants (November–December 2019) 25(6):1483–1495
foxtail [Setaria virdis (L.) Beauv.] and wild oat (Avena
fatua L.) and their effectiveness was studied using various
weed/crop densities (Boyetchko 1994, 1997). Literature
shows that no efforts were further made to understand the
mechanism involved in growth suppression of the wild oats
and no field trial or pot house studies were conducted. In
this study, rhizosphere bacteria belonging to Bacillus sia-
mensis and Bacillus endophyticus were characterized,
which suppressed the growth of wild oat under pot house
conditions. Moreover, seed bacterization of wheat and wild
oat with promising rhizobacterial isolates RWA52,
RWA69 and SYB101 caused significant reduction in RDW
and SDW of A. fatua, whereas inoculation of these rhi-
zobacterial isolates showed significant increase in RDW
and SDW of wheat plants under pot house conditions.
Materials and methods
Isolation of bacteria from rhizosphere of wheat
Soil samples were collected from the rhizosphere of wheat
grown in the field areas of Bawal (28°30 18.5100 N; 76°350
52.6000 E), Rewari (28°100 2700 N; 76°380 4400 E) and Hisar
districts (29°090 51.6600 N; 75°410 2.9400 E) after 60 and
90 days of plant growth. The soils of the Hisar farm were
sandy loam in texture (organic carbon, 0.44%; EC, 0.42;
pH, 7.2, available N, P and K, 105, 12 and 186 kg ha-1,
respectively), whereas soil of the Bawal region were loamy
sand in texture (organic carbon, 0.42%; EC, 0.24; pH, 7.9,
available N, P and K, 112, 21 and 334 kg ha-1, respec-
tively). Dilution plating of composite rhizosphere soil
samples was made on Luria–Bertani (LB) medium plates.
After 3–4 days incubation of plates at 28 ± 2 °C, rhi-
zobacterial colonies were selected based on typical mor-
phological and pigment producing characteristics. A total
of 88 rhizobacterial isolates selected from rhizosphere soil
were transferred on LB agar medium slopes (Sindhu et al.
1999). These selected isolates were screened for growth
retardation effects on weed and plant growth-promoting
attributes for wheat crop. Bacillus subtilis strain SYB101
with bioherbicidal effect on management of Phalaris minor
weed was used as reference strain (Phour and Sindhu
2018).
Identification of promising bacterial isolates
Preliminary characteristics of the promising isolates
included colony morphology, Gram staining, physiological
and biochemical tests. Two best performing rhizobacterial
isolates were then identified using 16S ribosomal RNA
(rRNA) gene sequencing. Total genomic DNA was
extracted using a DNA isolation kit and the 16S rRNA
Physiol Mol Biol Plants (November–December 2019) 25(6):1483–1495
gene was amplified by polymerase chain reaction (PCR)
using the universal primers 27F 50 (AGA GTT TGA TCC
TGG CTC AG) 30 and 1492R 50 (TAC GGY TAC CTT
GTT ACG ACT T) 30 , which are designed to amplify
universally conserved regions of the 16S rRNA gene and
resulted in the amplification of an approximately 1500 base
pair PCR product. Amplification and purification of the
product was done as described previously (Pandey et al.
2002). The phylogenetic tree was constructed using Clus-
talX, DAMBE and Mega7 software.
Screening of rhizobacterial isolates for production
of indole acetic acid and d-aminolevulinic acid
All the 88 rhizobacterial isolates were screened for in vitro
antagonistic and herbicidal activities, and thirty isolates
were finally evaluated for plant growth promoting benefi-
cial activities. For IAA estimation, selected bacterial cul-
tures were inoculated (in triplicate) in 30 ml LB broth
flasks supplemented with DL-tryptophan @ 100 lg ml-1
and were incubated at 28 ± 2 °C for 72 h under stationary
conditions of growth. Culture samples were centrifuged at
10,000 rpm for 15 min (Remi Instruments, Mumbai, India)
and IAA was determined in the culture supernatant by the
method as described by Malik and Sindhu (2011).
For ALA determination, selected antagonistic bacterial
cultures were inoculated (in triplicate) in 10 ml LB broth
tubes supplemented with 15 mM glycine and succinate.
Culture samples were withdrawn after incubation at
28 ± 2 °C for 48 h and centrifuged at 10,000 rpm for
15 min (Remi Instruments, Mumbai, India). ALA was
determined in the culture supernatant by the method as
described by Mauzerall and Granick (1955).
Screening of rhizobacterial isolates for utilization
of 1-aminocyclopropane-1-carboxylate
Minimal medium (Dworkin and Foster 1958) supple-
mented with 3 mM ACC was used for ACC utilization
(Penrose and Glick 2003). 48 h old growth of rhizobacte-
rial isolate was spotted on medium plates and growth was
recorded after 5 days of incubation at 28 ± 2 °C. The
bacterial isolates showing good growth on ACC supple-
mented medium plates, indicating high efficiency of ACC
utilization as nitrogen source. Minimal medium plates
containing ammonium sulfate as nitrogen source were used
as control for growth comparison of different bacterial
isolates.
1485
Efficacy of rhizobacterial inoculation on growth
of weed and wheat
Selected rhizobacterial isolates were checked for suppres-
sion of growth of Avena fatua weed and for growth stim-
ulation effects on growth of wheat (Triticum aestivum)
variety WH1105 under pot house conditions. Seeds of
wheat/weed were inoculated with 10 ml culture suspension
of bacteria (107–108 cells ml-1 of growth suspension).
Inoculated seeds were sown in pots (with three replications
for each treatment) and uninoculated seeds were sown as
control (Chaudhary and Sindhu 2016). The soil used for
growth of plants in greenhouse study was collected from
Ludas village (Hisar district) from two to ten cm depth of
soil after harvesting of the pearl millet crop. The soil used
was sandy soil in texture with the following characteristics
i.e., organic carbon, 0.26%; electrical conductivity,
0.48 dSm-1; pH, 7.8; available phosphorus, 10 kg ha-1,
available K2O, 228 kg ha-1; available nitrogen,
78 kg ha-1. Soil was supplemented with required doses of
nitrogen and phosphorus fertilizers. After germination,
eight healthy seedlings were maintained in each pot. The
observations for root and shoot growth of wheat and weeds
were recorded at 25, 50 and 75 days of plant growth.
Statistical analysis
Completely Randomized Design (CRD) was used for
experimental data analysis. All determinations in the
experiment were carried out in triplicate and data repre-
sented are average values of three replications. The C.D.
values represent coefficient of deviation.
Results
Microbial communities inhabiting the rhizosphere of dif-
ferent crop plants have been found to suppress/inhibit the
growth of weeds by reducing seed germination, weed
density and biomass (Park et al. 2015; Sindhu and Sehra-
wat 2017). In this study, 30 rhizobacterial isolates were
finally selected, out of 88 rhizobacterial isolates initially
obtained, for plant growth promoting beneficial activities.
Based on preliminary screening in relation to in vitro
antagonistic and herbicidal activities, five rhizobacterial
isolates were studied for growth suppression of wild oat
weed under pot house conditions and the possible mecha-
nism of weed growth inhibition was explored.
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In vitro screening for beneficial attributes 6% 3%

10%
of rhizobacteria 36% Very low (3-6 μg/ml)
Low (6-12 μg/ml)
Thirty selected rhizobacterial isolates were tested for pro- Medium (12-18 μg/ml)
duction of indole acetic acid, d-aminolevulinic acid and 45%
High (> 18 μg/ml)
utilization of 1-aminocyclopropane-1-carboxylate. Four Very high (> 30 μg/ml)
isolates i.e., BWA1, RWA55 and RWA72 produced 16.73,
16.24 and 12.12 lg ml-1 IAA, respectively (Table 1).
Maximum production of IAA was observed in isolate Fig. 1 Percent frequency of selected rhizobacterial isolates for IAA
RWA52 (19.18 lg ml-1), whereas isolate BWA18 pro- production
duced the highest amount of IAA (53.80 lg ml-1). Only
nineteen percent isolates showed medium to high amount RWA48, RWA63, SYB101 and HMM97 showed more
of IAA production (Fig. 1). Ten bacterial isolates i.e., than 5 lg ml-1 ALA production (Table 1; Fig. 2). Other
BWA1, BWA6, BWA19, BWA25, BWA27, BWA36, nineteen bacterial isolates showed less production of ALA
(\ 5 lg ml-1). Screening of rhizobacterial isolates for

Table 1 Plant growth promoting characteristics of selected rhizobacterial isolates

S. No. Rhizobacterial isolates IAA concentration (lg ml-1) ALA concentration (lg ml-1) Category of ACC utilizing isolates

1. BWA1 16.73 5.70 ?

2. BWA2 3.49 4.29 ???
3. BWA6 4.07 5.11 ??
4. BWA7 10.85 4.58 -
5. BWA8 6.21 4.16 ?
6. BWA14 6.15 4.33 ????
7. BWA18 53.80 4.79 ?
8. BWA19 3.55 5.00 ???
9. BWA20 4.52 4.37 ?????
10. BWA23 6.75 3.70 ?????
11. BWA25 6.78 7.12 ??
12. BWA27 12.00 5.70 ?
13. BWA29 6.12 4.70 ?????
14. BWA36 5.33 5.04 ?
15. BWA38 6.81 4.79 ?????
16. BWA40 6.96 4.29 ????
17. RWA42 7.78 4.37 -
18. RWA48 4.52 5.79 -
19. RWA52 19.18 4.58 ??
20. RWA53 4.52 4.74 -
21. RWA55 16.24 4.62 ?
22. RWA59 9.74 4.54 ?
23. RWA63 7.60 5.41 ?
24. RWA69 4.76 4.95 ????
25. RWA71 3.89 4.08 ??
26. RWA72 12.12 4.87 ???
27. SYB101 8.62 9.14 ??
28. HMM97 7.66 5.58 -
29. HMM8 5.39 4.95 ????
30. SB153 5.42 3.87 ??
Growth of rhizobacterial isolates on minimal medium supplemented with ACC is denoted as: ?: less, ??: moderate, ???: good, ????: very
good, ?????: significant growth and, -: no growth

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conditions. At 25 days of growth, inoculation with bacte-

33% rial isolate RWA69 caused 121 and 140% increase in RDW
and SDW of wheat growth, respectively and its inoculation
Low (< 5 μg/ml)
67%
decreased the root dry weight of A. fatua by 11% and also
Medium (> 5μg/ml)
decreased the shoot dry weight of the weed (Table 2;
Fig. 5). Increase in root dry weight (171%) and shoot dry
weight (110%) of wheat plants was observed by inocula-
tion of Bacillus isolate SYB101, whereas its inoculation
Fig. 2 Percent frequency of selected rhizobacterial isolates for decreased RDW and SDW of A. fatua (Table 2; Fig. 6). At
production of d-aminolevulinic acid
50 days of observation, inoculation with bacterial isolates
RWA69 and SYB101 caused significant increase in RDW
AAC utilization showed that only four isolates i.e.,
and SDW of wheat growth, whereas their inoculation
BWA20, BWA23, BWA29 and BWA38 showed signifi-
decreased RDW and SDW of A. fatua (Table 2; Figs. 7, 8).
cant growth on ACC supplemented plates (Table 1).
Inoculation with bacterial isolate RWA52 showed 48 and
Fourteen rhizobacterial isolates showed little growth,
12% gain in root and shoot dry weight shoot of wheat
whereas seven isolates showed moderate growth on ACC
growth, respectively, whereas its inoculation improved the
supplemented plates. Five isolates lacked the ability to
root dry weight by 24% but decreased the shoot dry weight
utilize ACC and did not grow on ACC supplemented
of A. fatua by 12% (Table 2).
plates. Overall, ACC utilization ability was observed with
Inoculation of rhizobacterial isolate BWA18 decreased
only 52.94% rhizobacterial isolates (Figs. 3, 4).
the root dry weight by 9% but showed 4% increase in SDW
of wheat at 75 days of observation. Whereas, its inocula-
Inoculation effect of selected rhizobacteria
tion decreased the root dry weight (14%) of weed and
on growth of wheat and weed
minor increase (2%) was observed in shoot dry weight of
A. fatua in comparison to RDF amended uninoculated soil
Five rhizobacterial isolates i.e., BWA18, BWA29,
treatment (Table 2; Fig. 9). Inoculation of bacterial isolate
RWA52, RWA69 and SYB101 were finally selected for
RWA52 resulted in 31 and 70% gain in root and shoot dry
seed bacterization of wheat and weed, based on the vari-
weight of wheat, respectively, whereas its inoculation
ation in their beneficial attributes, under pot house
decreased the RDW and SDW of A. fatua weed by 32 and
15%, respectively (Fig. 10). Similarly, inoculation with
13% 17% rhizobacterial isolate RWA69 showed 38% gain in root dry
13% Nil weight and shoot dry weight of wheat was improved by
Very low 131%, and its inoculation caused 16 and 13% decrease in
10% 27% Low RDW and SDW of A. fatua, respectively. Inoculation with
Medium another promising rhizobacterial isolate SYB101 showed
High 79 and 150% increase in RDW and SDW of wheat,
20%
Very high respectively and its inoculation decreased the root dry
weight by 19% and caused 15% decrease in shoot dry
weight of A. fatua.
Fig. 3 Percent frequency of rhizobacterial isolates for ACC
utilization

Fig. 4 Growth of different

rhizobacterial isolates on
minimal medium plates and on
medium supplemented with
ammonium sulphate and ACC

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Table 2 Inoculation effect of rhizobacterial isolates on plant biomass of wheat and weed at different stages of plant growth
Treatments Days of Wheat Weed
growth
RDW (g SDW (g RDW (g SDW (g
plant-1) plant-1) plant-1) plant-1)

Control (uninoculated soil) ? Wheat 25 0.14 0.19 – –

50 0.21 0.52 – –
75 0.53 0.76 – –
Control (uninoculated soil ? N, P 25 0.28 0.40 – –
fertilizers) ? Wheat
50 0.46 0.68 – –
75 0.75 0.94 – –
T1 ? Weed (Avena fatua) 25 – – 0.03 0.06
50 – – 0.07 0.22
75 – – 0.12 0.41
T2 ? Weed (Avena fatua) 25 – – 0.18 0.28
50 – – 0.34 0.64
75 – – 0.62 1.04
T1 ? Wheat ? Weed 25 0.18 0.18 0.05 0.14
50 0.30 0.46 0.12 0.30
75 0.60 0.68 0.18 0.36
T2 ? Wheat ? Weed 25 0.32 0.36 0.14 0.30
50 0.48 0.64 0.29 0.52
75 0.72 0.83 0.44 0.84
T2 ? Wheat ? BWA18 25 0.38 0.46 – –
50 0.50 0.72 – –
75 0.68 0.98 – –
T2 ? Weed ? BWA18 25 – – 0.22 0.34
50 – – 0.38 0.74
75 – – 0.53 1.06
T2 ? Wheat ? Weed ? BWA18 25 0.30 0.32 0.15 0.28
50 0.42 0.64 0.25 0.52
75 0.62 1.32 0.39 0.68
T2 ? Wheat ? BWA29 25 0.36 0.50 – –
50 0.48 0.82 – –
75 0.58 1.60 – –
T2 ? Weed ? BWA29 25 – – 0.36 0.62
50 – – 0.74 1.12
75 – – 1.02 1.42
T2 ? Wheat ? Weed ? BWA29 25 0.22 0.36 0.22 0.40
50 0.40 0.64 0.52 0.84
75 0.64 1.20 0.74 1.08
T2 ? Wheat ? RWA52 25 0.40 0.50 – –
50 0.68 0.76 – –
75 0.98 1.60 – –
T2 ? Weed ? RWA52 25 – – 0.20 0.30
50 – – 0.42 0.56
75 – – 0.67 0.88
T2 ? Wheat ? Weed ? RWA52 25 0.32 0.32 0.16 0.22
50 0.62 0.64 0.30 0.38
75 0.76 1.36 0.42 0.54
T2 ? Wheat ? RWA69 25 0.62 0.96 – –

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Table 2 continued
Treatments Days of Wheat Weed
growth
RDW (g SDW (g RDW (g SDW (g
plant-1) plant-1) plant-1) plant-1)

50 0.86 1.22 – –
75 1.04 2.18 – –
T2 ? Weed ? RWA69 25 – – 0.21 0.42
50 – – 0.30 0.56
75 – – 0.52 0.90
T2 ? Wheat ? Weed ? RWA69 25 0.52 0.82 0.10 0.22
50 0.74 1.04 0.25 0.44
75 0.92 1.86 0.58 0.72
T2 ? Wheat ? SYB101 25 0.76 0.84 – –
50 1.02 1.52 – –
75 1.34 2.35 – –
T2 ? Weed ? SYB101 25 – – 0.20 0.20
50 – – 0.28 0.52
75 – – 0.50 0.88
T2 ? Wheat ? Weed ? SYB101 25 0.58 0.68 0.11 0.24
50 0.94 1.40 0.22 0.46
75 1.16 2.56 0.36 0.74
CD at 5% 25 0.032 0.030 0.040 0.042
50 0.030 0.038 0.041 0.036
75 0.040 0.184 0.037 0.182
Treatment T1 represents uninoculated soil, whereas T2 represents uninoculated soil amended with N and P fertilizer doses. Data represented are
average values of three replications. The C.D. values represent coefficient of deviation. RDW root dry weight, SDW shoot dry weight

1 TACGGYTACCTTGTTACGACTT-30 ). Genomic DNA of

rhizobacterial isolates was obtained according to the
0.8 method described by Pitcher et al. (1989). The partial 16S
Wheat shoot dry
0.6
weight rDNA gene sequence of the RWA52 and RWA69 bacterial
isolates were then used as a query to search for similar
g/plant

Avena fatua shoot

dry weight
0.4 sequences in GenBank database. The rhizobacterial isolates
Wheat root dry
weight RWA52 and RWA69 were identified as Bacillus siamensis
0.2
Avena fatua root dry and Bacillus endophyticus, respectively (Figs. 11, 12).
weight
0

Fig. 5 Inoculation effect of bacterial isolates on biomass of wheat
and Avena fatua at 25 days of plant growth
Identification of promising rhizobacterial isolates
by the 16S rRNA sequencing
Two promising rhizobacterial isolates RWA52 and
RWA69 were identified by using universal primers 8-27F
(50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -
Discussion
Besides climate change and abiotic stresses, harmful phy-
topathogens, insects and weeds are major constraints to
enhance food productivity. Recently, significant efforts are
being made worldwide to characterize effective biocontrol
agents for control of plant diseases as well as for sup-
pressing the growth of weeds (Kennedy and Stubbs 2007;
Mejri et al. 2010; Park et al. 2015; Sindhu et al. 2016). The
production of phytotoxins, phytohormones (IAA, ALA)
and antibiotics by rhizosphere bacteria residing in the

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Fig. 6 Growth of wheat and weed (A. fatua) plants after inoculation Control (uninoculated soil ? N, P Fertilizers) ? Weed; T28: T2 ?
with bacterial isolate SYB101 at 25 days of plant growth. T1: Control Wheat ? SYB101; T29: T2 ? Weed ? SYB101; T30:
(uninoculated soil) ? Wheat; T2: Control (uninoculated soil ? N, P T2 ? Wheat ? Weed ? SYB101
Fertilizers) ? Wheat; T3: Control (uninoculated soil) ? Weed; T4:

vicinity of the weed roots have been implicated in exerting

1.6 deleterious effects on the growth of weeds. Interestingly,
1.4 many rhizospheric bacteria showing weed growth inhibit-
1.2 Wheat shoot dry
ing properties (Kremer and Kennedy 1996; Sindhu et al.
1 weight 2018), were found to stimulate the growth of crop plants
g/plant

0.8 Avena fatua shoot dry (Li and Kremer 2006; Mejri et al. 2010). Production of
weight
0.6 phytohormones and other secondary metabolites have been
Wheat root dry
0.4 weight reported as the predominant mechanisms of biocontrol of
0.2 Avena fatua root dry weeds/phytopathogens (Saraf et al. 2014; Sindhu et al.
0 weight 2018; Phour and Sindhu 2019).
In this study, 30 rhizobacterial isolates (selected out of
total 88 rhizobacterial isolates) were screened for produc-
Fig. 7 Inoculation effect of different bacterial isolates on biomass of tion of IAA, ALA and ACC utilization ability. Maximum
wheat and Avena fatua at 50 days of plant growth production of IAA was observed in bacterial isolate

Fig. 8 Growth of wheat and weed (A. fatua) plants after inoculation Control (uninoculated soil ? N, P Fertilizers) ? Weed; T25: T2 ?
with bacterial isolate RWA69 at 50 days of plant growth. T1: Control Wheat ? RWA69; T26: T2 ? Weed ? RWA69; T27:
(uninoculated soil) ? Wheat; T2: Control (uninoculated soil ? N, P T2 ? Wheat ? Weed ? RWA69
Fertilizers) ? Wheat; T3: Control (uninoculated soil) ? Weed; T4:

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2.5
2
Wheat shoot dry
1.5
weight
g/plant
Avena fatua shoot
dry weight
1 sion of weed great brome and stimulation of the growth of
Wheat root dry
weight
0.5
Avena fatua root dry
0 weight
Fig. 9 Inoculation effect of different rhizobacterial isolates on
biomass of wheat and Avena fatua at 75 days of plant growth
BWA18 (53.80 lg ml-1) followed by isolate RWA52
([ 18 lg ml-1) (Table 1). Bacterial isolate RWA52
exhibiting significant production of IAA significantly
inhibited the growth of Avena fatua, whereas its inocula-
tion significantly improved the wheat growth at different
stages of observations (Table 2). Similar suppressive effect
on the growth of weed morning glory was correlated with
the IAA production by Bradyrhizobium japonicum isolate
GD3 (Kim and Kremer 2005). Sachdev et al. (2009) iso-
lated nine Klebsiella pneumoniae strains from rhizosphere
of wheat and six strains showed variable amounts of IAA
production. Seed bacterization with these six strains sig-
nificantly increased root length and shoot height of inoc-
ulated wheat seedlings over the uninoculated control plants
under pot house studies. Meliani et al. (2017) reported that
Fig. 10 Growth of wheat and weed (A. fatua) plants after inoculation
with bacterial isolate RWA52 at 75 days of plant growth. T1: Control
(uninoculated soil) ? Wheat; T2: Control (uninoculated soil ? N, P
Fertilizers) ? Wheat; T3: Control (uninoculated soil) ? Weed; T4:
1491
plant growth promoting bacteria Pseudomonas fluorescens
and P. putida produced high levels of IAA i.e., 89 and
116 lg ml-1, respectively and significantly enhanced the
plant growth. Similarly, the amount of IAA produced by P.
trivialis strain X33d was correlated with growth suppres-
durum wheat (Mejri et al. 2010).
ALA has been reported to act as herbicide and growth
promoting factor in agriculture applications and it also
contribute towards tolerance of salt and cold temperature in
plants (Watanabe et al. 2000). In this study, ten bacterial
isolates BWA1, BWA6, BWA19, BWA25, BWA27,
BWA36, RWA48, RWA63, HMM97 and SYB101 showed
more than 5 lg ml-1 ALA production (Table 1; Fig. 2).
Zhang et al. (2008) reported that the combined treatment of
oil seed rape plants with ALA and post emergence herbi-
cide, propyl 4-(2-(4,6-dimethoxypyrimidine-2-yloxy ben-
zylamino) benzoate (ZJ0273) improved the growth and
development of oilseed rape seedlings (Brassica napus cv.
ZS758) and also improved weed control efficacy. Khan-
delwal et al. (2018) observed that 80% of the rhizobacterial
isolates produced ALA. Out of these isolates, four rhi-
zobacterial isolates showed root growth inhibition of
Chenopodium album weed at both 5th and 10th day of seed
germination. Phour and Sindhu (2019) reported that
Bacillus flexus strain JMM24 produced 11.70 lg ml-1 of
ALA and its inoculation on Lathyrus aphaca weed caused
92% decrease in RDW and SDW at 75 days of growth
under pot house conditions.
Some rhizobacterial strains possessing ACC deaminase
activity have recently been found to improve the biomass
Control (uninoculated soil ? N, P Fertilizers) ? Weed; T22: T2 ?
Wheat ? RWA52; T23: T2 ? Weed ? RWA52; T24:
T2 ? Wheat ? Weed ? RWA52
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1492
Fig. 11 Phylogenetic (neighbour joining) tree based on 16S rRNA sequence analysis indicating phylogenetic position of bacterial isolate
RWA52
and yield of different crops (Glick 2003; Chaudhary and
Sindhu 2017). Out of 30 isolates tested in this study,
52.94% rhizobacterial isolates showed ACC utilization
ability (Table 1; Fig. 3). B. endophyticus strain RWA69,
which showed significant growth on ACC supplemented
plates, showed significant decrease in the RDW and SDW
of weed and promoted the growth of wheat at 50 days of
plant growth (Fig. 8). Stearns et al. (2012) reported that the
ACC deaminase-containing bacterium Pseudomonas
putida strain UW4 significantly promoted the growth of
Brassica napus. Magnucka and Pietr (2015) selected three
fluorescent pseudomonads containing ACC deaminase
activity from rhizosphere of wheat (Triticum aestivum L.)
and rape (Brassica napus L.), and Pseudomonas brassi-
cacearum strain RZ310 posessing highest ACC deaminase
activity showed beneficial effect on the growth of wheat
seedlings even at low cell densities. Ten bacterial strains
belonging to Enterobacter, Serratia, Klebsiella and
Escherichia genera were isolated (Carlos et al. 2016) and
Serratia strain showed the positive correlation between
ACC deaminase and IAA production in the presence of
heavy metals, and its inoculation also promoted the growth
of Helianthus annuus.
In this study, four rhizobacterial isolates BWA18,
RWA52, RWA69 and SYB101 stimulated the growth of
wheat, and rhizobacterial isolates i.e., BWA18, BWA29
123
Physiol Mol Biol Plants (November–December 2019) 25(6):1483–1495
and RWA52 inhibited the growth of A. fatua in comparison
to RDF amended uninoculated soil treatments (Table 2;
Figs. 5, 6, 7, 8, 9, 10). Rhizobacterial isolates having
inhibitory effect on the growth of weed A. fatua and
growth-promoting effect on wheat could further be tested
in controlled experimental designs in particular soil type,
plant species and the environmental factors. In earlier
studies, plant genotypes/varieties have been found to affect
the colonization ability of the inoculated strains on the
roots (Sheng 2005; Phour and Sindhu 2018). Therefore, the
selection of competitive and effective bacterial strains is
desirable among the indigenous soil bacteria, which could
easily adapt and establish in the particular soil conditions at
the inoculation site (Paau 1989; Sindhu and Dadarwal
2000). In similar studies, Kennedy et al. (1991) screened
1000 pseudomonads isolates for differential inhibition of
downy brome (Bromus tectorum) and winter wheat. Out of
these isolates, bacteria free culture filtrates obtained from
only 8% isolates showed inhibitory effect on the root
growth of downy brome weed, but no effect was observed
on the root growth of winter wheat on agar. When these
isolates were inoculated under field conditions, only two
isolates (0.2%) suppressed the growth of downy brome
by * 31–53% whereas, this treatment showed * 18–35%
increase in the yield of winter wheat. Similarly, Li and
Kremer (2006) reported that inoculation of Pseudomonas
Physiol Mol Biol Plants (November–December 2019) 25(6):1483–1495
Fig. 12 Phylogenetic (neighbour joining) tree based on 16S rRNA sequence analysis indicating phylogenetic position of bacterial isolate
RWA69
fluorescens strain G2-11 onto wheat and soybean crops
suppressed the growth of Ipomea sp. and Convolvolus
arvensis weeds, while its inoculation promoted the growth
of wheat and soybean crops. Amara et al. (2015) reported
significant increase in length of root and shoot as well as
dry weight of root and shoot in the wheat crop in com-
parison to uninoculated control by coinoculation of Psy-
chrobacter meritimus sp. strain A18, Serratia
proteamaculans sp. and Bacillus anthracis sp. strain A29,
along with nitrogenous and phosphatic fertilizers under pot
and field conditions. These results showed that inoculation
of rhizobacterial strains may influence the resource parti-
tioning towards the growth suppression of weed and pro-
motion of wheat ultimately leading to improved crop
productivity in sustainable agriculture.
Conclusions
Until now, increase in wheat production using high yield-
ing varieties has provided sufficient crop yield to keep pace
with ever-increasing world population. Recently, the
application of chemical fertilizers even at maximum dose is
not further increasing the yield of various crops. To further
improve wheat productivity, promising rhizosphere
1493
bacteria could be used for growth stimulation of wheat
along with their suppressive effect on growth of phy-
topathogens and weeds. In this study, bacterial isolate
BWA18 with high IAA (53.80 lg ml-1) production ability
showed growth inhibition of weed and stimulated the
growth of wheat at 25, 50 and 75 days of observation.
Another isolate RWA52 (with IAA production,
19.18 lg ml-1) showed growth stimulation of wheat and
growth inhibition of weed as compared to RDF amended
uninoculated soil treatment. Another bacterial isolate
RWA69 selected on the basis of ACC utilization ability,
showed maximum growth stimulation of wheat at 25, 50
and 75 days of observation. Two promising rhizobacterial
isolates RWA52 and RWA69 were identified by the 16S
rRNA sequence analysis as Bacillus siamensis and Bacillus
endophyticus, respectively. Results suggested that rhi-
zobacterial isolates with growth inhibiting properties of A.
fatua weed and growth promoting properties of wheat
could be further explored under field conditions. The use of
promising rhizobacterial isolates as bioherbicides will be
more useful in the fields, where herbicide resistant popu-
lation of A. fatua exists. Thus, development of bioherbi-
cides is need-based ecofriendly and cost effective
technology for pollution free sustainable agriculture.
123
1494
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