Wang et al.

BMC Microbiology (2020) 20:38

https://doi.org/10.1186/s12866-020-1708-z

RESEARCH ARTICLE Open Access

Beneficial bacteria activate nutrients and

promote wheat growth under conditions of
reduced fertilizer application
Juanjuan Wang2†, Ruochen Li2†, Hui Zhang2, Gehong Wei1,2 and Zhefei Li2*

Abstract
Background: Excessive application of chemical fertilizer has exerted a great threat to soil quality and the
environment. The inoculation of plants with plant-growth-promoting rhizobacteria (PGPR) has emerged as a great
prospect for ecosystem recovery. The aim of this work to isolate PGPRs and highlights the effect of bacterial
inoculants on available N/P/K content in soil and on the growth of wheat under conditions of reduced fertilizer
application.
Results: Thirty-nine PGPRs were isolated and tested for their growth-promoting potential. Thirteen isolates had
nitrogen fixation ability, of which N9 (Azotobacter chroococcum) had the highest acetylene reduction activity of
156.26 nmol/gh. Eleven isolates had efficient phosphate solubilizing ability, of which P5 (Klebsiella variicola) released
the most available phosphorus in liquid medium (231.68 mg/L). Fifteen isolates had efficient potassium solubilizing
ability, of which K13 (Rhizobium larrymoorei) released the most available potassium in liquid medium (224.66 mg/L).
In culture medium supplemented with tryptophan, P9 (Klebsiella pneumoniae) produced the greatest amount of
IAA. Inoculation with the bacterial combination K14 + 176 + P9 + N8 + P5 increased the alkali-hydrolysed nitrogen,
available phosphorus and available potassium in the soil by 49.46, 99.51 and 19.38%, respectively, and enhanced
the N, P, and K content of wheat by 97.7, 96.4 and 42.1%, respectively. Moreover, reducing fertilizer application by
25% did not decrease the available nitrogen, phosphorus, and potassium in the soil and N/P/K content, plant
height, and dry weight of wheat.
Conclusions: The bacterial combination K14 + 176 + P9 + N8 + P5 is superior candidates for biofertilizers that may
reduce chemical fertilizer application without influencing the normal growth of wheat.
Keywords: Plant growth-promoting rhizobacteria, Phosphate-solubilizing, Potassium-solubilizing, Nitrogen fixation,
Promote

Background and consumer of chemical fertilizers, accounting for one-

China has the largest population in the world.The appli- third of the global total consumption [2]. For example,
cation of chemical fertilizer has played a key role in in- from 1997 to 2006, the average application of nitrogen
creasing grain production in China to produce sufficient fertilizer for rice increased from 145 kg/ha− 1 to 300 kg/
food for a very large population with limited arable land ha− 1 in the Taihu region [3], a significantly larger increase
[1]. In recent decades, to meet the nutritional needs of a than in countries outside China. However, the utilization
rapidly growing population, China’s the annual total de- efficiency of these fertilizers is only 30–40%, indicating
mand for chemical fertilizers has shown an upward that most applied chemical fertilizer is lost by different
trend; that country has become the largest manufacturer pathways, such as ammonia (NH3) volatilization and
leaching [4–6].
* Correspondence: lizhefei@hotmail.com Repeated overuse of chemical fertilizer can have a
†
Juanjuan Wang and Ruochen Li contributed equally to this work. negative effect on soil quality and soil microbial commu-
2
Shaanxi Key Laboratory of Agricultural and Environmental Microbiology,
College of Life Science, Northwest A&F University, Yangling, Shaanxi, China
nity structure. Excessive fertilizer aggravates the decline
Full list of author information is available at the end of the article of soil organic matter and fertility and accelerates soil
© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wang et al. BMC Microbiology (2020) 20:38 Page 2 of 12

acidification, which in turn reduces crop yield [7, 8]. solubilization, plant growth promotion and reduction of
Therefore, there is an urgent need to find an environ- the need for fertilizer in wheat. This study was planned
mentally friendly strategy to reduce the application of to (1) isolate PGPR from the rhizosphere/roots of wheat
chemical fertilizer and increase crop yield. In many and screen them in vitro for plant growth promotion
countries, it is a widely accepted practice to enhance potential; (2) investigate the effect of PGPR on the
sustainable agricultural production by inoculating crops availability of nitrogen, phosphorus and potassium in
with plant growth-promoting rhizobacteria (PGPR). soil and the growth of wheat; and (3) evaluate the role of
Plant-growth-promoting rhizobacteria are free-living PGPR inoculation in reducing fertilizer input.
soil microorganisms that inhabit the rhizosphere or
plant roots during plant growth and development. PGPR Results
can promote plant growth, help prevent pathogen inva- Plant-growth-promoting potential
sion and improve plant adaptability to abiotic or bio- Among the bacterial isolates in this study, thirteen
logical stresses [9–11]. The beneficial effects of PGPR on strains (N1-N13) were able to grow on nitrogen-free
plants can be explained by different mechanisms, includ- medium. An acetylene reduction activity (ARA) assay in-
ing (1) nitrogen fixation [12]; (2) inorganic phosphate dicated that all thirteen isolates had nitrogenase activity,
solubilization and organic phosphate mineralization [13]; ranging from 15.14 to 156.26 nmol/gh. The nitrogenase
(3) production of plant growth regulators or phytohor- activity of isolates N2 and N9 was significantly higher
mones such as indole-3-acetic acid (IAA), cytokinins, than that of all other strains, and their ARA values were
and gibberellins [14, 15]; (4) production of siderophores, 113.67 and 156.26 nmol/gh, respectively (Fig. 1a).
1-amino-cyclopropane-1 -carboxylate (ACC) deaminase, Eleven bacterial isolates were able to form a halo zone
and hydrogen cyanate [16, 17]; (5) and biological control around the colonies by P solubilization. Four isolates (P2,
of phytopathogens and insects by synthesizing antibiotics P4, P6 and P7) could dissolve inorganic but not organic
or fungicidal compounds or competiting with detrimen- phosphate, three isolates (P8, P10 and P11) could dissolve
tal microorganisms [18, 19]. organic but not inorganic phosphate and four isolates (P1,
Agricultural problems caused by the long-term use of P3, P5 and P9) were able to dissolve both inorganic and or-
pesticides, fertilizers and other products have become in- ganic phosphate (Table 1). P5 had the highest solubilization
creasingly prominent. This not only pollutes agricultural index (2.56) on PVK medium, while P11 had the highest
products but also causes an imbalance in the propor- solubilization index (4.57) on organic phosphorus dissol-
tions of various nutrients, the destruction of organic ution medium (Fig. 1b, c and Table 1). Quantitative deter-
matter in the soil and a decrease in the structural integ- mination showed that the range of inorganic phosphate
rity and properties of aggregates, leading to soil compac- solubilization varied from 67.18 to 231.68 mg/L, and the
tion, salinization and disease aggravation. However, soil range of organic phosphate solubilization was between
microorganisms are involved in various biotic activities of 13.67 and 40.40 mg/L (Table 1).
the soil ecosystem to promote dynamic turnover and sus- Fifteen bacterial isolates were able to form clear zone
tainable crop production. Thus PGPR and their interac- on Aleksandrov medium. After inoculating these 15
tions with plants have great application prospects in strains of bacteria, the soluble potassium content in the
ecological agriculture and sustainable agriculture. Co- supernatant of culture medium ranged from 38.55 to
inoculation of cotton with Azotobacter chroococcum 224.66 mg/L. K13 strain had the strongest potassium re-
strains can positively influence plant growth and reduce leasing ability (Table 2). Moreover, among 39 plant
nitrogen fertilization doses by 50% [20]. Korir et al. [21] growth promotion bacteria, except isolates P6, P10 and
conducted experiments using a low-phosphorous soil K15, the others could produced IAA in the presence of
under greenhouse conditions to examine the effect of tryptophan. But the amount of IAA produced by them is
PGPR on the nodulation and growth of common bean. quite different. P9 produced maximum IAA followed by
The results showed that mixed inoculaiton with strains of P5, K7, K5, P1 and K13 (Tables 1, 2, 3). Of all the iso-
Bacillus megaterium and Rhizobium tropici significantly lates, only K14 had strong cellulose degradation ability
increased nodule fresh weight, plant dry weight, and root (Additional file 3: Table S1).
dry weight by 192.2, 124.5, and 126.7%, respectively.
In northwest China, wheat is a staple food that has Identification of bacteria
special regional importance. Low annual precipitation Thirty-nine rhizosphere/endophytic bacteria with poten-
limits crop growth, and there is much emphasis on the tial growth-promoting ability were isolated in vitro. Based
use of large amounts of chemical fertilizer to increase on the sequencing results of the 16S rRNA gene, these
wheat yields. However, information is scarce regarding bacterial isolates belonged to 15 genera (Additional file 3:
the isolation of PGPR from the wheat rhizosphere and Table S1). The isolate N9 (GenBank accession number:
the role of PGPR in nitrogen fixation, phosphate MN658515) had the closest genetic relationship with
Wang et al. BMC Microbiology (2020) 20:38 Page 3 of 12

Fig. 1 Plant growth promoting potential of bacteria. a Acetylene reduction activity (ARA) of thirteen isolates. N1: Paenibacillus SP. N2, N4, N7:
Azotobacter sp. N3, N5: Rheinheimera sp. N6: Agrobacterium sp. N8, N11: Pseudomonas sp. N9: Azotobacter chroococcum N10: Enterobacter sp. N12:
Pantoea sp. N13: Erwinia sp. b Solubilization zone of organic phosphate c Solubilization zone of inorganic phosphate

Table 1 Phosphorus releasing ability of PGPR

Isolates PGPR activities Inorganic phosphate Organic phosphate IAA pH
production
SI P content(mg/L) SI P content (mg/L)
P 1 (Klebsilla pneumoniae) Organic and inorganic P solubilizing, IAA production 1.73 172.28 ± 3.15 + ++ 5.32
P 2 (Klebsiella sp.) Inorganic P solubilizing, IAA production 1.52 109.25 ± 3.15 – ++ 5.46
P 3 (Enterobacter asburiae) Organic and inorganic P solubilizing, IAA production 2.38 189.19 ± 3.91 + + 5.13
P 4 (Raoultella sp.) Inorganic P solubilizing, IAA production 1.17 95.79 ± 3.78 – + 5.78
P 5 (Klebsiella variicola) Organic and inorganic P solubilizing, IAA production 2.56 231.68 ± 3.13 + +++ 4.96
P 6 (Agrobacterium sp.) Inorganic P solubilizing 1.32 67.18 ± 3.74 – – 6.32
P 7 (Rhizobium sp.) Inorganic P solubilizing, IAA production 1.89 140.21 ± 3.81 – + 5.96
P 8 (Enterobacter sp.) Organic P solubilizing, IAA production – 2.87 13.64 ± 1.00 + 6.78
P 9 (Klebsiella sp.) Organic and inorganic P solubilizing, IAA production + 2.50 29.93 ± 2.47 +++ 5.63
P 10 (Enterobacter sp.) Organic P solubilizing – 2.00 8.49 ± 0. 7 – 6.37
P 11 (Comamonas SP.) Organic P solubilizing, IAA production – 4.57 40.40 ± 1.90 + 5.26
Control – 1.23 ± 0.12 – 0.67 ± 0.07 – 7.02
Note: + means the isolate can solubilize organic, inorganic phosphate or produce IAA, − means the isolate can not solubilize organic/inorganic phosphate
Wang et al. BMC Microbiology (2020) 20:38 Page 4 of 12

Table 2 Potassium releasing ability of PGPR

Isolates PGPR activities SI K content(mg/L) IAA production pH
K1 (Klebsiella variicola) K and P solubilizing, IAA production 2.78 95.97 ± 3.75 ++ 4.49
K2 (Enterobacter sp.) K and P solubilizing, IAA production 2.86 94.64 ± 3.00 + 4.66
K3 (Raoultella sp.) K and P solubilizing, IAA production 2.79 83.80 ± 2.40 + 4.97
K4 (Klebsiella variicola) K and P solubilizing, IAA production 2.65 57.20 ± 1.62 ++ 6.11
K5 (Klebsiella variicola) K and P solubilizing, IAA production 1.39 64.16 ± 2.19 ++ 5.15
K6 (Klebsilla pneumoniae) K and P solubilizing, IAA production 1.57 38.55 ± 2.82 ++ 6.32
K7 (Klebsiella sp.) K and P solubilizing, IAA production 1.26 39.71 ± 1.23 +++ 6.18
K8 (Raoultella sp.) K and P solubilizing, IAA production 2.02 67.36 ± 2.55 + 5.29
K9 (Raoultella sp.) K and P solubilizing, IAA production 1.88 49.84 ± 2.77 + 5.03
K10 (Pseudomonas sp.) K and P solubilizing, IAA production 3.59 193.33 ± 3.87 + 4.42
K11 (Advenella sp.) K and P solubilizing, IAA production 2.97 117.20 ± 3.50 + 4.78
K12 (Agrobacterium sp.) K and P solubilizing, IAA production 3.02 154.78 ± 3.11 + 4.31
K13 (Rhizobium sp.) K and P solubilizing, IAA production 3.85 224.66 ± 2.69 ++ 4.03
K14 (Bacillus sp.) K and P solubilizing, IAA production 1.93 81.13± 3.50 + 5.22
K15 (Sphinqomonas sp.) K and P solubilizing 9.92 104.59 ± 2.74 – 5.45
Control – – 31.96 ± 1.60 – 7.20

Azotobacter chroococcum IAM12666, and its 16S rDNA tumefaciens CCNWGS0286) preserved in our laboratory
sequence homology was 99.93%. The isolate N8 (Acces- were inoculated into plant roots. However, there was no
sion number: MN700634) showed 97.88% similarity with difference in wheat plant height among 13 single-
Pseudomonas furukawaii KF707. The strain P9 (Accession inoculated treatments and the non-inoculated treatment.
number: NR_036794) had 100% similarity with Klebsiella Therefore, the isolates with nitrogen fixation, inorganic/or-
pneumoniae ATCC13884. The isolate K14 (Accession ganic phosphate and potassium-releasing ability and strain
number: MN704638) had 99.34% similarity with Bacillus 176 were combined. Ten out of thirty-six PGPRs combina-
niacini IFO 15566. The isolates P5 (Accession number: tions significantly increased the plant height of wheat.
MN658477), K10 (Accession number: MN704376), and Meanwhile, eight combinations enhanced the dry weight of
K13 (Accession number: MN662624) had the closest gen- wheat (Fig. 2). K14 (Bacillus sp.) + 176 (Agrobacterium
etic relationship with Klebsiella variicola, Pseudomonas tumefaciens) + P9 (Klebsiella pneumoniae) + N8 (Pseudo-
migulae, and Rhizobium larrymoorei in the phylogenetic monas sp.) + P5 (Klebsiella variicola) and K14 (Bacillus
tree based on 16S rRNA gene sequences reconstructed sp.) + 176 (Agrobacterium tumefaciens) + P9 (Klebsiella
using MEGA (version 6), respectively (Additional file 1: pneumoniae) + N9 (Azotobacter chroococcum) + K10
Figure S1). (Pseudomonas sp.) maximized the dry weight and height of
wheat. Shoot height and plant dry mass were increased by
Plant growth parameters 18.59 and 105% (P < 0.05), respectively, in plants inoculated
To evaluate the growth-promoting effect of PGPR on with K14 + 176 + P9 + N8 + P5 and K14 + 176 + P9 + N9 +
wheat, 12 isolates with strong growth-promoting ability and K10 group compared to plants without inoculum (Fig. 2
one growth-promoting bacterium “176” (Agrobacterium and Additional file 3: Table S2).

Table 3 PGPR activities of nitrogen fixing bacteria

Isolates PGPR activities IAA Isolates PGPR activities IAA
N1 (Paenibacillus SP.) Nitrogen fixation, IAA production + N8 (Pseudomonas sp.) Nitrogen fixation, IAA production +
N2 (Azotobacter sp.) Nitrogen fixation, IAA production + N9 (Azotobacter chroococcum) Nitrogen fixation, IAA production +
N3 (Rheinheimera sp.) Nitrogen fixation, IAA production + N10 (Enterobacter sp.) Nitrogen fixation, IAA production ++
N4 (Azotobacter sp.) Nitrogen fixation, IAA production + N11 (Pseudomonas sp.) Nitrogen fixation, IAA production +
N5 (Rheinheimera sp.) Nitrogen fixation, IAA production + N12 (Pantoea sp.) Nitrogen fixation, IAA production ++
N6 (Agrobacterium sp.) Nitrogen fixation, IAA production + N13 (Erwinia sp.) Nitrogen fixation, IAA production +
N7 (Azotobacter sp.) Nitrogen fixation, IAA production +
Wang et al. BMC Microbiology (2020) 20:38 Page 5 of 12

Fig. 2 The effect of bacterial combination on a plant height and b plant dry biomass of wheat. Values are means ± SE. abcd letters on the bars
denote differences on the basis of a t-test (p < 0.05). C0: Control C1: K14 + 176 + P9 + N8 + K10 C6: K14 + 176 + P9 + N8 + P5 C7: K14 + 176 + P9 +
N9 + K10 C9: K14 + 176 + P9 + N9 + K13 C12: K14 + 176 + P9 + N9 + P5 C15: K14 + 176 + P9 + N10 + K13 C16: K14 + 176 + P9 + N10 + P1 C24: K14 +
176 + P11 + N8 + P5 C33: K14 + 176 + P11 + N10 + K13 C34: K14 + 176 + P11 + N10 + P1

Determination of nutrients in rhizosphere soil N/P/K uptake were observed when the soil was inocu-
Ten bacterial combinations with good growth-promoting lated with different combinations of bacteria compared
effects were inoculated into wheat roots, and the available with the non-inoculated control. For wheat inoculated
nitrogen, phosphorus and potassium contents in the soil with different bacterial combinations, the plant N con-
were determined after 80 days of plant growth. Compared tent increased from 40.7 to 97.7% (P < 0.05). Addition-
with the control, the alkali-hydrolysed nitrogen, available ally, the P content of wheat increased from 41.2 to
phosphorus and available potassium in all inoculated soil 96.4%, and the K content increased from 2.3 to 42.1%.
were increased to varying degrees. Among the combina-
tions K14 (Bacillus sp.) + 176 (Agrobacterium tumefa- Effect of reducing fertilizer application
ciens) + P9 (Klebsiella pneumoniae) + N8 (Pseudomonas Since the combination of K14 + 176 + P9 + N8 + P5 could
sp.) + P5 (Klebsiella variicola) had the most notable effect activate nutrients in soil and promote the utilization of
on soil nutrient improvement. The alkali-hydrolysed ni- nitrogen, phosphorus and potassium by plants, we vali-
trogen, available phosphorus and available potassium in- dated whether this PGPR combination inoculation could
creased by 49.46, 99.51 and 19.38%, respectively (Table 4). reduce fertilizer doses. The results are shown in Figs. 4
and 5, Compared with a 100% fertilizer dose, the reduc-
Determination of N/P/K content in wheat tion of fertilizer application by 25, 50 and 100% signifi-
In order to verify whether inoculation treatment can cantly inhibited plant growth. When no fertilizer was
promote nutrient uptake by plants, the contents of nitro- added to the soil, the combined PGPR inoculation sig-
gen, phosphorus and potassium in wheat were deter- nificantly increased plant height, tiller counts, fresh
mined. As shown in Fig. 3, significant increases in plant weight and dry weight but still did not perform as well
Wang et al. BMC Microbiology (2020) 20:38 Page 6 of 12

Table 4 The effect of bacterial combination on available N/P/K content in soil

Combination Alkali-hydrolyzed nitrogen (mg/kg soil) Available P (mg/kg soil) Available K (mg/kg soil)
c
C0 Control 12.13 ± 0.2 7.48 ± 0.8c 87.47 ± 2.1d
b
C1 K14 + 176 + P9 + N8 + K10 18.71 ± 1.1 11.69 ± 1.4b 95.79 ± 2.9bcd
a
C6 K14 + 176 + P9 + N8 + P5 21.98 ± 1.4 14.92 ± 1.1a 104.42 ± 2.8a
b ab
C7 K14 + 176 + P9 + N9 + K10 19.28 ± 1.5 13.09 ± 0.4 98.24 ± 2.8abc
b
C9 K14 + 176 + P9 + N9 + K13 19.55 ± 0.9 14.19 ± 1.1ab 100.51 ± 1.0ab
b b
C12 K14 + 176 + P9 + N9 + P5 18.71 ± 0.8 11.49 ± 1.0 90.38 ± 6.3cd
b
C15 K14 + 176 + P9 + N10 + K13 18.01 ± 1.4 12.00 ± 1.2ab 93.86 ± 4.4bcd
b ab
C16 K14 + 176 + P9 + N10 + P1 18.13 ± 0.9 12.63 ± 1.1 89.20 ± 5.2cd
c b
C24 K14 + 176 + P11 + N8 + P5 14.51 ± 1.6 11.47 ± 1.2 91.33 ± 1.5bcd
c b
C33 K14 + 176 + P11 + N10 + K13 14.76 ± 0.6 11.18 ± 0.9 86.51 ± 2.5d
c b
C34 K14 + 176 + P11 + N10 + P1 14.51 ± 1.6 11.72 ± 0.9 93.27 ± 4.5bcd
Values are means ± SE. abcd letters on the bars denote differences on the basis of a t-test (p < 0.05)

as 100% fertilizer application without PGPR. However, In sum, these results showed that the combination of
there was no difference in plant height, fresh weight, K14 + 176 + P9 + N8 + P5 could promote nitrogen, phos-
tiller counts, or N/P/K content between PGPR com- phorus and potassium accumulation in wheat, and re-
bination + 75% fertilizer and 100% fertilizer without duce the application of chemical fertilizer by 25%.
PGPR (Figs. 4 and 5). Compared with 100% fertilizer,
PGPR combination + 75% fertilizer did not reduce Discussion
available nitrogen, phosphorus or potassium in the There is a very complex system of interactions among mi-
soil (Additional file 2: Figure S2). croorganisms and between microorganisms and plants in

Fig. 3 Promotion of PGPRs combination on a nirogen, b phosphorus, and c potassium uptake for wheat. Values are means ± SE. abcde letters on
the bars denote differences on the basis of a t-test (p < 0.05)
Wang et al. BMC Microbiology (2020) 20:38 Page 7 of 12

Fig. 4 The effect of bacterial combination K14 + 176 + P9 + N8 + P5 on a plant height, b plant fresh weight, c plant dry weight, d N content in plant, e P
content in plant, and f K content in plant of wheat. T1: without fertilizer, T2: PGPR combination 6, T4: 50% fertilizer + PGPR combination 6, T6: 75% fertilizer
+ PGPR combination 6, T7: 100% fertilizer. Values are means ± SE. abcd letters on the bars denote differences on the basis of a t-test (p < 0.05)

soil. Under certain conditions, plants recruit beneficial mi- stresses [23], inhibit the growth of plant pathogens by pro-
croorganisms to colonize the rhizosphere or root tissue by ducing antimicrobial compounds or competing with patho-
secreting metabolites to the soil [22]. These microorgan- gens for nutrients [24], and activate nutrients in soil [25].
isms can improve plant adaption to biotic and abiotic The nitrogen-fixing, and P/K-releasing potential of bacteria
Wang et al. BMC Microbiology (2020) 20:38 Page 8 of 12

Fig. 5 a The effect of bacterial combination K14 + 176 + P9 + N8 + P5 and fertilizer application on tiller number of potted wheat. Values are
means ± SE. abcd letters on the bars denote differences on the basis of a t-test (p < 0.05). b Phenotype of wheat under different fertilization
treatments. T1: without fertilizer, T2: PGPR combination 6, T3: 50% fertilizer, T4: 50% fertilizer + PGPR combination 6, T5: 75% fertilizer, T6: 75%
fertilizer + PGPR combination 6, T7: 100% fertilizer

isolated from the rhizosphere and root of wheat was ana- to plants. Some soil microorganisms can dissolve other-
lysed. Finally, thirty-nine bacterial strains with plant- wise insoluble phosphate and potassium. Several studies
growth-promoting ability were isolated using different have shown that different bacterial genera, such as
screening media. The 16S rRNA sequence analysis revealed Pseudomonas, Bacillus, Rhizobium, Agrobacterium, Kleb-
that these bacteria belong to 15 genera (Additional file 3: silla, and Erwinia, have the potential to solubilize and
Table S1). The nitrogenase activity of Azotobacter was release phosphorus/potassium from soil [28]. In the
much higher than that of other bacteria (Fig. 1), and strain present work, 11 phosphate-solubilizing bacteria and 15
N9, with the highest nitrogenase activity, showed 99.93% potassium-solubilizing bacteria were isolated from the
similarity with A. chroococcum IAM12666. The results pre- rhizosphere and roots of wheat. These microbial isolates
sented in this paper are in agreement with previous works. showed potassium solubilization index from 1.26 to 3.85
A. chroococcum has been isolated from the rhizosphere or or phosphate solubilization indices from 1.17 to 4.57 on
roots of many plants such as litchi, rice, and faba bean [26, PVK or Aleksandrov medium (Tables 1 and 2). These
27]. The results of a study by Rodelas et al. [27] showed that bacterial isolates comprise 9 Klebsilla, 4 Enterobacter, 4
the acetylene reduction activity of Azotobacter sp. was be- Raoultella, 2 Rhizobium, 2 Agrobacterium, 1 Bacillus, 1
tween 9.7 and 257.7 nmol C2H4 h− 1 vial− 1, while A. chroo- Advenella, 1 Microbacterium, 1 Comamonas, and 1
coccum isolates had a high capacity for acetylene reduction. Pseudomonas isolate (Additional file 3: Table S1), this is
In addition to nitrogen, phosphorus and potassium are consistent with previous studies. Maliha et al. [29]
the other two essential plant growth-limiting nutrients. showed that bacteria released organic acids and de-
Although potassium and phosphorus are abundant ele- creased the pH of the rhizosphere, which led to the dis-
ments in soil, they are mostly bound to other minerals. solution of the mineral phosphate. The selected bacterial
Approximately 95–99% of phosphorous and potassium strains were grown using a broth culture method, and
are present in insoluble form and therefore unavailable the phosphorus/potassium content and pH of the
Wang et al. BMC Microbiology (2020) 20:38 Page 9 of 12

culture medium were analysed quantitatively. Compared and ATCR1_07569 (monooxygenase) involved in IAM
with the non-inoculated control, the inoculated treat- (indole-3-acetamide) pathway. Previous study has con-
ment showed significantly increased phosphorus/potas- firmed that excessive production of IAA by strain 176 is
sium content in broth and reduced median pH. an important reason for plant growth-promoting [33]. In
Meanwhile, the more pH decreased, the larger the K. variicola DX120E, the genome contains nif gene clus-
solubilization index and the more phosphorus/potassium ter, indole-3-pyruvate decarboxylase, and siderophore
content in broth was observed. Potassium-solubilizing enterobactin synthesis genes (entABCDEF) which con-
bacteria can also dissolve tricalcium phosphate in tribute to N2 fixation, indole-3-acetic acid production,
medium (Tables 1 and 2 and Additional file 3: Table siderophore production, and phosphate solubilization.
S1). The results indicated that the bacteria we isolated Besides ferric iron uptake transcriptional regulator gene
dissolve insoluble phosphate/potassium mainly by pro- fur and indole-3-glycerol phosphate synthase gene trpC,
ducing organic acids. there are 10 inorganic phosphorus dissolving genes
In addition to focusing on biological nitrogen fixation (from pqqA to pqqF) in P. furukawaii KF707T genome.
and phosphate/potassium solubilization, we investigated This strain also encodes antioxidant enzymes, such as
the IAA-producing potential of in these bacterial iso- superoxide dismutase and catalase, which have the abil-
lates. The results showed that most of the isolates could ity to remove free radicals under stresses. The genome
synthesize IAA, but their production rates were quite of B. niacini NBRC 15566T contains 13 genes involved
different. This variation is due to the different synthetic in iron transport and ferric uptake regulation, and there
pathways, key genes and regulatory strategies of different are trpC gene (encoding indole-3-glycerol phosphate
bacteria [30]. Bacteria isolated from the rhizosphere of synthase) and 4 phospholipase genes. Thus these
soybean and rice have been shown to produce 10.54 to growth-promoting genes may be closely related to the
84.72 mg/kg IAA [31, 32]. The strain P9 (P-solubilizing increase of available N, P, K in soil and plant growth
bacteria) showed the highest IAA production, followed (Fig. 1, Tables 1, 2, and 3). Thus we speculated that the
by the strains P5 and K7 (Tables 1, 2, and 3). The mea- improvement of plant growth can be attributed to nitro-
sured production of IAA was higher in this work than in gen fixation and phosphate/potassium solubilization by
previous reports. This suggested that these isolates could PGPR, which provides great deal of nutrition for plant
serve as efficient biofertilizer candidates for activating development [34]. Furthermore, IAA can be produced
phosphorus/potassium and simultaneously promoting by inoculated PGPR. IAA is involved in cell division,
crop growth. root growth, and stem elongation, increasing the surface
It is important to consider that the growth-promoting area of roots such that plants con obtain additional
performance of bacteria will be influenced by biotic and water and nutrients [35]. A large number of studies have
abiotic factors in soil. We combined bacteria with differ- focused on the role of PGPR in promoting crop growth
ent functions and performed experiments in a green- [36, 37]. However, bacteria are not a complete substitute
house. The results of pot-based experiments showed for fertilizers. In the present study, the potential of
that ten combinations significantly increased the avail- PGPR in reducing fertilizer application was also investi-
able phosphorus/potassium and alkali hydrolysed nitro- gated. The plant dry weight, N/P/K content and tiller
gen in the soil, and the plant height, dry biomass, and counts were not affected by a 25% decrease in fertilizer
N/P/K content of wheat also increased after inoculation after inoculation with the combination K14 + 176 + P9 +
with these ten bacterial combinations. Among them, the N8 + P5. In conclusion, these PGPR can be used as bio-
combination K14 + 176 + P9 + N8 + P5 had the best fertilizer and have broad application prospects in sus-
growth-promoting effect (Fig. 4). In combination K14 + tainable agricultural development.
176 + P9 + N8 + P5 with better growth promoting effect,
the isolates P5, N8, P9 and K14 had the closest genetic Conclusions
relationship with Klebsiella Klebsiella variicola, Pseudo- These results clearly showed that N2-fixing, P-solubilizing,
monas furukawaii, Klebsiella pneumoniae and Bacillus K-solubilizing and IAA-producing bacteria can signifi-
niacini, respectively. Through analysis the genome of the cantly increase available nitrogen, phosphorus and potas-
strain 176, two possible pathways for IAA synthesizing sium in soil. A combination of growth-promoting bacteria
have been found. The genes ATCR1_06021 and with different functions effectively improved N/P/K up-
ATCR1_10873, ATCR1_02965, ATCR1_10883, ATCR1_ take and plant growth in wheat. These bacterial isolates
10878, ATCR1_02970, and ATCR1_23021 may be ino- allow the use of lower chemical fertilizer doses than trad-
volve in the synthesis of tryptophan. Genes ATCR1_ itional fertilization strategies and have greater application
17572 (encoding amine oxidase) and ATCR1_06371 (in- potential in the field. However, future studies are needed
dole-3-acetaldehyde dehydrogenase) involved in TAM to investigate other conditions, locations and crops to ver-
(tryptamine) pathway, while ATCR1_17003 (nitrilase) ify the reliability of the present study.
Wang et al. BMC Microbiology (2020) 20:38 Page 10 of 12

Methods China), Potassium aluminium silicate 3.0 g (Daiquan

Isolation of bacteria Fine Chemical & Technology Co., Ltd., China), agar 15 g
Samples of wheat were obtained from fields in which and distilled water 1000 ml], respectively. The plates
wheat had been cultivated continuously for more than were incubated at 28 °C for 7 days and observed for the
10 years in Yangling, China. Plants with rhizosphere soil clear zone around the colonies. The solubilization index
were placed in sterile plastic bags and brought back to (SI) was calculated as follows:
the laboratory. For isolation of rhizosphere bacteria, 1 g
of rhizospheric soil was taken in 10 ml of sterile water
SI ¼ Diameter of zone of clearance=Diameter of colony
and shaked for 10 min. 1 g of fresh wheat roots were
washed under running tap water and surface sterilized
with 4% NaOCl for 1 min. After washing six times with Then, the phosphate/potassium-soluble strains were
sterilized distilled water, the roots were ground with a inoculated into the corresponding liquid medium and
sterilized mortar and pestle. These two suspension were incubated at 28 °C, 180 rpm for 5 days. The pH value of
then serially diluted to 10− 8 grades, 0.1 ml of each dilu- solution was measured by PHS-3C pH meter (INESA
tion of 1 × 10− 6, 1 × 10− 7, and 1 × 10− 8 dilution was Scientific Instrument Co., Ltd, China).The P concentra-
spreaded on TY agar plates (Tryptone 5 g, Yeast extract tion in the solution was determined using the molybdo-
3 g, CaCl2 0.2 g, Agar 15 g, distilled water 1000 ml). The vanadate method [40]. The K concentration in the
plates were incubated at 28C for 5 days. Isolates showing solution was estimated using atomic absorption spec-
different colony characters were selected, purified and trometry [41].
stored in liquid TY medium containing 20% glycerol at
− 80 °C. Production of IAA
Bacterial isolates were grown in TY supplemented with
Screening for plant-growth-promoting traits tryptophan (500 mg/L) and incubated at 28 °C for 48 h
Nitrogen fixation on a rotary shaker. Cultures were centrifuged at 12,000 g
The purified colonies were streaked on Ashby agar (su- for 10 min. A 1 ml volume of supernatant was placed
crose 10 g, KH2PO4 0.2 g, NaCl 0.2 g, MgSO4 0.2 g, into an EP tube, and an equal volume of Salkowski re-
K2SO4 0.2 g, CaCO3 5 g, agar 15 g, distilled water 1000 agent was added and the optical density of 530 nm
ml) and incubated at 30 °C for 7 days. The sticky col- (OD530) of solution was measured after 30 min of reac-
onies were inoculated into Ashby solid medium vials tion in dark. The different concentration of IAA (0, 20,
and incubated at 28 °C for 24 h. Five hundred microliters 40, 60, 80, 100, 120, 140, 160 and 180 mg/L) was pre-
of air was replaced with 500 μL of acetylene, and the pared. OD530 of each concentration IAA solution was
vials were incubated at 28 °C for 24 h. Then 600 μL of determined and the standard curve was drawn. Then the
gas was extracted from vials and the ethylene content IAA content of bacterial culture medium was calculated
was analysed with a gas chromatograph (Shimadzu GC- based on standard curve.
17A, Japan) equipped with an H-flame ionization de-
tector using methods described by Hardy et al. [38]. At Determination of cellulose degradation
the same time, the bacteria on surface of the solid Each bacterial colone was inoculated on cellulolytic cul-
medium in the vials were washed down, dried and ture medium (sodium carboxymethyl cellulose 5 g, yeast
weighed. The acetylene reducing activity (ARA) was cal- extract 2 g, KH2PO4, 0.5 g MgSO4 0.5 g, agar 15 g and
culated as follows: distilled water 1000 ml) and incubated at 28 °C for 3
days. Then, colonies were stained with 1 mg/mL Congo
Ethylene production ðnmolÞ red dye for 15 min and decolorized with 1 mol/L NaCl
ARA ¼
Bacteria weight ðg Þ  Reaction tiem ðhÞ for 30 min. Colonies with clear zones indicated that the
bacteria had the ability to degrade cellulose.

Solubilization of phosphate and potassium Molecular characterization of the PGPR strains

Each bacterial colony was inoculated on PVK medium The total genomic DNA of 39 bacterial strains was ex-
[39], organic phosphorus dissolution medium [glucose tracted according to the method of Wilson & Carson
10 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, FeSO4 0.03 [42]. The 16S rRNA region was amplified using forward
g, MnSO4 0.03 g, CaCO3 5 g, lecithin 0.2 g (Aladdin Bio- primer 27F (5-AGAGTTTGATCC TGGCTCAG-3) and
chemical Technology Co., Ltd., China), agar 15 g and reverse primer 1492R (5-TACGGCTACCTTGTTACG
distilled water 1000 ml], and Aleksandrov medium [glu- ACTT-3). The resulting products were confirmed on a
cose 5 g, MgSO4 0.5 g, CaCO3 0.1 g, FeCl3 0.006 g, 1% agarose gel, and then the correct PCR products were
Ca3PO4 2.0 g (Kemiou Chemical Reagent Co., Ltd., sequenced Sangon Biotech (Shanghai) Co., Ltd., China.
Wang et al. BMC Microbiology (2020) 20:38 Page 11 of 12

The obtained gene sequences were analysed using the months, and the fresh weight, dry weight, shoot length,
BLASTN program. and tiller count of the plants were determined, along
with the available N/P/K concentrations of the soil.
Plant growth promotion
The top layer of the soil, 0–20 cm deep, was collected Data analyses
from abandoned farmland, air dried, passed through a 2 All experiments in the present study were performed in
mm sieve and thoroughly homogenized. Pots measuring at least three replicates. The data were analysed using
23 cm in diameter and 17 cm in height were filled with SPSS software version 23.0 (Armonk, NY, USA). The
3 kg of soil each. Seeds of the wheat (Triticum aestivum significant differences among various treatments were
L.) variety Xiaoyan 22 were purchased from Yangling compared using Fisher’s protected LSD test with P ≤
Jinnuo seed Co., Ltd. in China. The seeds were surface 0.05.
sterilized with a 3% sodium hypochlorite solution for 10
mins, followed by six rinses with deionized distilled
Supplementary information
water. Sterilized wheat seeds were planted in the pots. Supplementary information accompanies this paper at https://doi.org/10.
After the wheat seedlings had been growing for 1 week, 1186/s12866-020-1708-z.
10 plants were retained in each pot, and 10 mL of a
combination of 5 PGPRs (contains 2 mL each PGPR Additional file 1: Figure S1. Neighbour-joining tree based on 16S rRNA
gene sequences of PGPRs.
with 108 colony formation unit) was inoculated into
Additional file 2: Figure S2. The effect of bacterial combination K14 +
plant roots. K14 has a strong ability to degrade cellulose, 176 + P9 + N8 + P5 and fertilizer application on N/P/K content in soil.
176 is a known growth-promoting bacterium preserved Values are means ± SE. abcd letters on the bars denote differences on
in our laboratory; all the PGPR combinations contain the basis of a t-test (p < 0.05).
these two bacterial strains. The other three strains were Additional file 3: Identification of bacteria and its promoting effect on
plants.
selected from phosphorus-solubilizing (P1, P5, P9 and
P11), nitrogen-fixing (N8, N9 and N10) and potassium-
solubilizing bacteria (K10 and K13). This results in 36 Abbreviations
ARA: Acetylene reduction activity; IAA: Indole-3-acetic acid; PGPR: Plant
inoculation combinations. Uninoculated pots were in- growth promoting rhizobacteria; SI: Solubilization index
cluded as negative controls. All pots were placed in a
greenhouse with a 16/8 h photoperiod (light/dark) and Acknowledgements
Not applicable.
at 25 ± 1 °C for 80 days. The plants were watered with
250 mL water every 3 days during the experiment. The Authors’ contributions
plants were harvested after 80 days, and the fresh weight, ZL, and GH make conception and design of this study. RL, JW and HZ
dry weight, shoot length, and N/P/K content of the conduct the experiments. ZL carry out the data analysis and manuscript
writing. All authors read and approved the final manuscript.
plants and the available N/P/K concentrations in the soil
were determined. Alkali-hydrolyzable N was analyzed by Funding
Kjeldahl digestion methods on a Kjeltec 8400 analyzer This work was supported by the National Key Research and Development
unit (Foss-Tecator AB, Hoganas, Sweden), available P in Program of China (2018YFD0200403) and natural science basic research plan
in shaanxi province of China (2018JM3004). The funders played no role in
plant samples and soils were estimated by molybdovana- the design of the study, analysis, and interpretation of data or in writing the
date method; whereas available K was analyzed using an manuscript.
atomic absorption spectrophotometer (Hitachi, Tokyo,
Japan). Availability of data and materials
The datasets supporting the conclusions of this article are included within
the article. All the bacterial strains can be obtained from the lab of Gehong
Experiments on reducing fertilizer application Wei upon request.
The soil was collected from farmland. Soil treatment,
PGPR inoculation and planting were the same as in the Ethics approval and consent to participate
Not applicable.
“Plant growth promotion” section. The experimental
treatment included pots with no fertilizer and PGPRs Consent for publication
mixture (T1); pots inoculated with PGPRs mixture (T2); Not applicable.
pots supplied with 50% (T3), 75% (T5) or 100% fertilizer
(T7); pots inoculated with PGPRs mixture and supplied Competing interests
The authors declare that they have no competing interests.
with 50% (T4) or 75% fertilizer (T6). The experiment
was set up with 8 replicate pots per treatment. The Author details
1
doses of chemical fertilizer were based on a dose com- State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F
University, Yangling, Shaanxi, China. 2Shaanxi Key Laboratory of Agricultural
monly used by farmers (105 kg N, 112.5 kg P2O5, and and Environmental Microbiology, College of Life Science, Northwest A&F
120 kg K2O per hectare). Plants were harvested after 3 University, Yangling, Shaanxi, China.
Wang et al. BMC Microbiology (2020) 20:38 Page 12 of 12

Received: 27 September 2019 Accepted: 17 January 2020 bacterial biofertilizer for cotton (Gossypium hirsutum): effect in reducing N
fertilization. Rev Argent Microbiol. 2017;49(4):377–83.
21. Korir H, Mungai NW, Thuita M, Hamba Y, Masso C. Co-inoculation effect of
rhizobia and plant growth promoting Rhizobacteria on common bean
References growth in a low phosphorus soil. Front Plant Sci. 2017;8:141.
1. Hou Y, Ma L, Gao ZL, Wang F, Sims JT, Ma W, Zhang F. The driving forces 22. Huang AC, Jiang T, Liu Y, Bai Y, Reed J, Qu B, Goossens A, Nutzmann H, Bai
for nitrogen and phosphorus flows in the food chain of China, 1980 to Y, Osbourn A. A specialized metabolic network selectively modulates
2010. J Environ Qual. 2013;42(4):962–71. Arabidopsis root microbiota. Science. 2019;364(6440):eaau6389.
2. Sun B, Zhang L, Yang L, Zhang F, Norse D, Zhu Z. Agricultural non-point 23. Ali S, Charles TC, Glick BR. Amelioration of high salinity stress damage by
source pollution in China: causes and mitigation measures. Ambio. 2012; plant growth-promoting bacterial endophytes that contain ACC deaminase.
41(4):370–9. Plant Physiol Bioch. 2014;80:160–7.
3. Zhao X, Zhou Y, Min J, Wang S, Shi W, Xing G. Nitrogen runoff dominates 24. Berendsen RL, Vismans G, Yu K, Song Y, De Jonge R, Burgman WP, Burmolle M,
water nitrogen pollution from rice-wheat rotation in the Taihu Lake region Herschend J, Bakker PAHM, Pieterse CMJ, Pieterse CM. Disease-induced
of China. Agric Ecosyst Environ. 2012;156:1–11. assemblage of a plant-beneficial bacterial consortium. ISME J. 2018;12(6):1496–507.
4. Anderson KR, Moore PA, Miller DM, Delaune PB, Edwards DR, Kleinman PJ, 25. Nakayan P, Hameed A, Singh S, Young L, Hung M, Young C. Phosphate-
Cademenun BJ. Phosphorus leaching from soil cores from a twenty-year solubilizing soil yeast Meyerozyma guilliermondii CC1 improves maize (Zea
study evaluating alum treatment of poultry litter. J Environ Qual. 2018;47(3): mays L.) productivity and minimizes requisite chemical fertilization. Plant
530–7. Soil. 2013;373:301–15.
5. Huang J, Xu C, Ridoutt BG, Wang X, Ren P. Nitrogen and phosphorus losses 26. Chennappa G, Adkarpurushothama CR, Suraj U, Tamilvendan K, Sreenivasa
and eutrophication potential associated with fertilizer application to MY. Pesticide tolerant Azotobacter isolates from paddy growing areas of
cropland in China. J Clean Prod. 2017;159:171–9. northern Karnataka, India. World J Microb Biot. 2014;30(1):1–7.
6. Wang X, Zou C, Gao X, Guan X, Zhang Y, Shi X, Chen X. Nitrate leaching 27. Rodelas B, Gonzalez-Lopez J, Pozo C, Salmeron V, Martinez-Toledo MV. Response
from open-field and greenhouse vegetable systems in China: a meta- of faba bean (Vicia faba L.) to combined inoculation with Azotobacter and
analysis. Environ Sci Pollut Res. 2018;25(31):31007–16. Rhizobium leguminosarum bv. viceae. Appl Soil Ecol. 1999;12(1):51–9.
7. Li XG, Jia B, Lv J, Ma Q, Kuzyakov Y, Li F. Nitrogen fertilization decreases the 28. Shrivastava M, Srivastava PC, D’Souza SF. Phosphate-solubilizing Microbes:
decomposition of soil organic matter and plant residues in planted soils. diversity and phosphates Solubilization mechanism. Singapore: Role of
Soil Biol Biochem. 2017;112:47–55. Rhizospheric Microbes in Soil. Springer; 2018. p. 137–65.
8. Qiao C, Xu B, Han Y, Wang J, Wang X, Liu L, Liu W, Wang S, Tan H, Liu Y, 29. Maliha R, Samina K, Najma A, Sadia A, Farooq L. Organic acids production
Zhao X. Synthetic nitrogen fertilizers alter the soil chemistry, production and and phosphate solubilization by phosphate solubilizing microorganisms
quality of tea. A meta-analysis. Agron Sustain Dev. 2018;38(1):10. under in vitro conditions. Pak J Biol Sci. 2004;7(2):187–96.
30. Li M, Guo R, Yu F, Chen XY, Zhao H, Li H, Wu J. Indole-3-acetic acid
9. Villarodriguez E, Parracota FI, Castrolongoria E, Lopezcervantes J,
biosynthesis pathways in the plant-beneficial bacterium Arthrobacter
Santosvillalobos SD. Bacillus subtilis TE3: a promising biological control
pascens ZZ21. Int J Mol Sci. 2018;19(2):443.
agent against Bipolaris sorokiniana, the causal agent of spot blotch in
31. Kim A, Shahzad R, Kang S, Seo C, Park Y, Park H, Lee I. IAA-producing
wheat (Triticum turgidum L. subsp. durum). Biol Control. 2019;132:135–43.
Klebsiella variicola AY13 reprograms soybean growth during flooding stress.
10. Cordero I, Balaguer L, Rincon A, Pueyo JJ. Inoculation of tomato plants with
J Crop Sci Biotechnol. 2017;20(4):235–42.
selected PGPR represents a feasible alternative to chemical fertilization
32. Bal HB, Das S, Dangar TK, Adhya TK. ACC deaminase and IAA producing
under salt stress. J Plant Nutr Soil Sci. 2018;181(5):694–703.
growth promoting bacteria from the rhizosphere soil of tropical rice plants.
11. Emami S, Alikhani HA, Pourbabaei AA, Etesami H, Sarmadian F,
J Basic Microb. 2013;53(12):972–84.
Motessharezadeh B. Effect of rhizospheric and endophytic bacteria with
33. Hao X, Xie P, Johnstone L, Miller SJ, Rensing C, Wei G. Genome sequence
multiple plant growth promoting traits on wheat growth. Environ Sci Pollut
and mutational analysis of plant-growth-promoting bacterium
Res. 2019;26(19):19804–13.
Agrobacterium tumefaciens CCNWGS0286 isolated from a zinc-Lead mine
12. Lin L, Li Z, Hu C, Zhang X, Chang S, Yang L, An Q. Plant growth-promoting
tailing. Appl Environ Microbiol. 2012;78(15):5384–94.
nitrogen-fixing Enterobacteria are in association with sugarcane plants
34. Etesami H, Alikhani HA. Evaluation of gram-positive rhizosphere and
growing in Guangxi, China. Microbes Environ. 2012;27(4):391–8.
endophytic bacteria for biological control of fungal rice (Oryzia sativa L.)
13. Zaheer A, Malik A, Sher A, Qaisrani MM, Mehmood A, Khan SU, Ashraf MU,
pathogens. Eur J Plant Pathol. 2017;147(1):7–14.
Mirza Z, Karim S, Rasool M. Isolation, characterization, and effect of
35. Zhang Z, Zhou W, Li H. The role of GA, IAA and BAP in the regulation of
phosphate-zinc-solubilizing bacterial strains on chickpea (Cicer arietinum L.)
in vitro shoot growth and microtuberization in potato. Acta Physiol Plant.
growth. Saudi J Biol Sci. 2019;26(5):1061–7.
2005;27(3):363–9.
14. Matsuda R, Handayani ML, Sasaki H, Takechi K, Takano H, Takio S.
36. Salvo LP, Cellucci GC, Carlino ME, De Salamone IE. Plant growth-promoting
Production of indoleacetic acid by strains of the epiphytic bacteria
rhizobacteria inoculation and nitrogen fertilization increase maize (Zea mays
Neptunomonas spp. isolated from the red alga Pyropia yezoensis and the
L.) grain yield and modified rhizosphere microbial communities. Appl Soil
seagrass Zostera marina. Arch Microbiol. 2018;200(2):255–65.
Ecol. 2018;6:113–20.
15. Cappellari LD, Santoro MV, Schmidt A, Gershenzon J, Banchio E. Induction
37. Robledoburitica J, Aristizaballoaiza JC, Ceballosaguirre N, Cabracendales T.
of essential oil production in Mentha x piperita by plant growth promoting
Influence of plant growth-promoting rhizobacteria (PGPR) on blackberry
bacteria was correlated with an increase in jasmonate and salicylate levels
(Rubus glaucus Benth. cv. Thornless) growth under semi-cover and field
and a higher density of glandular trichomes. Plant Physiol Bioch. 2019;141:
conditions. Acta Agronómica. 2018;67(2):258–63.
142–53.
38. Hardy RW, Holsten RD, Jackson EK, Burns RC. The acetylene-ethylene assay for
16. Sulochana MB, Jayachandra SY, Kumar SA, Parameshwar A, Reddy KM,
N2 fixation: laboratory and field evaluation. Plant Physiol. 1968;43(8):1185–207.
Dayanand A. Siderophore as a potential plant growth-promoting agent
39. Nautiyal CS. An efficient microbiological growth medium for screening
produced by Pseudomonas aeruginosa JAS-25. Appl Biochem Biotech. 2014;
phosphate solubilizing microorganisms. FEMS Microbiol Lett. 1999;170(1):265–70.
174(1):297–308.
40. King EJ. The colorimetric determination of phosphorus. Biochem J. 1932;
17. Glick BR. Bacteria with ACC deaminase can promote plant growth and help 26(2):292–7.
to feed the world. Microbiol Res. 2014;169(1):30–9. 41. Setiawati TC, Mutmainnah L. Solubilization of potassium containing mineral by
18. Sun G, Yao T, Feng C, Chen L, Li J, Wang L. Identification and biocontrol microorganisms from sugarcane Rhizosphere. Agric Agric Sci Proc. 2016;9:108–17.
potential of antagonistic bacteria strains against Sclerotinia sclerotiorum and 42. Wilson T, Carson J. Rapid, high-throughput extraction of bacterial genomic DNA
their growth-promoting effects on Brassica napus. Biol Control. 2017;104:35–43. from selective-enrichment culture media. Lett Appl Microbiol. 2001;32(5):326–30.
19. Etesami H, Emami S, Alikhani HA. Potassium solubilizing bacteria (KSB):
mechanisms, promotion of plant growth, and future prospects a review. J
Soil Sci Plant Nut. 2017;17(4):897–911. Publisher’s Note
20. Romeroperdomo F, Abril J, Camelo M, Morenogalvan A, Pastrana I, Springer Nature remains neutral with regard to jurisdictional claims in
Rojastapias DF, Bonilla R. Azotobacter chroococcum as a potentially useful published maps and institutional affiliations.