Microbiological Research 202 (2017) 4350

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Eect of succinate on phosphate solubilization in nitrogen xing bacteria MARK

harbouring chick pea and their eect on plant growth

Bhagya Iyera, Mahendrapal Singh Rajputa,b, Shalini Rajkumara,
a
Institute of Science, Nirma University, Ahmedabad, Gujarat, India
b
Department of Microbiology, Faculty of Science, Marwadi University, Rajkot, Gujarat, India

A R T I C L E I N F O A B S T R A C T

Keywords: Diverse nitrogen xing bacteria harbouring chick pea rhizosphere and root nodules were tested for multiple
Plant growth promoting rhizobacteria plant growth promoting traits like tricalcium phosphate (TCP) and rock phosphate (RP) solubilization, pro-
Phosphate solubilizing bacteria duction of ammonia, indole 3-acetic acid, chitinase, phytase and alkaline phosphatase. Isolates belonged to
Succinate mediated repression diverse genus like Enterobacter, Acinetobacter, Erwinia, Pseudomonas, Rhizobium, Sinorhizobium, Ensifer, Klebsiella,
Inorganic P solubilization
etc. Most isolates solubilized TCP and RP along with the lowering of media pH, indicating acidication to be the
chief mechanism behind this solubilization. However, lowering of media pH and P release decreased by
32100% when media was supplemented with succinate, a major component of plant root exudates indicating
succinate mediated repression of P solubilization. Maximum TCP and RP solubilization with P release of 850 g/
mL and 2088 g/mL was obtained with lowering of media pH up to 2.8 and 3.3 for isolate E43 and PSB1
respectively. This pH drop changed to 4.4 and 4.8 with 80% and 87% decrease in P solubilization in the presence
of succinate. Maximum 246 g/mL indole 3-acetic acid production in Lh3, 44.8 U/mL chitinase activity in MB3,
11.3 U/mL phytase activity in I91 and 9.4 U/mL alkaline phosphatase activity in SM1 were also obtained. Most
isolates showed multiple PGP traits which resulted in signicant plant growth promotion of chick pea plants.
Present study shows repression of P solubilization by succinate for various bacterial groups which might be one
of the reasons why phosphate solubilizing bacteria which perform well in vitro often fail in vivo. Studying this
repression mechanism might be critical in understanding the in vivo ecacy.

1. Introduction
Rhizosphere is a narrow region of soil in immediate proximity to the
root system inhabited by a large group of benecial bacteria called as
plant growth promoting rhizobacteria (PGPR) which inuence plant
growth through diverse direct and indirect mechanisms. Direct me-
chanisms of plant growth promotion (PGP) encompass nitrogen (N2)
xation, phosphate (P) solubilization, improving plant nutrient uptake,
siderophore production, modulating levels of phytohormones like in-
dole-3 acetic acid (IAA) whilst production of antibiotics, lytic enzymes,
ability to survive competition and inducing systemic resistance in
plants comprise the indirect mechanisms (Ahemad and Kibret, 2014). P
is an important macronutrient which occurs in soil in organic and in-
organic forms. However, most of the soil P is in a form unavailable for
plant uptake, but can be converted to plant available forms by phos-
phate solubilizing bacteria (PSB) (Zaidi et al., 2009) like Flavobacterium,
Achromobacter, Agrobacterium, Aerobacter, Bacillus, Erwinia, Micrococcus,
Pseudomonas, Burkholderia, Rhizobium, etc (Rodriguez and Fraga, 1999).
In addition to providing P to the plant, PSBs augment N2 xation,
improves availability of trace elements and secrete chemicals which aid
plant growth (Zaidi et al., 2009). Ability of bacteria to solubilize di-
calcium phosphate (DCP), tricalcium phosphate (TCP), rock phosphate
(RP) and hydroxyapatite have been studied (Shashidhar and Podile,
2010). Rock phosphates in the form of apatite, hydroxyapatite and
oxyapatite along with mineral phosphate in association with metals
form the biggest inorganic soil P reserve. This is followed by organic
phosphates in the form of phyate which comprises 50% of soil organic P
(McLaughlin et al., 1990). Major mechanism of inorganic P solubiliza-
tion is organic acid production leading to acidication. Gluconic acid is
an important organic acid produced by uorescent Pseudomonas (Vyas
and Gulati, 2009), P. cepacia (Goldstein et al., 1993), E. herbicola (Liu
et al., 1992), etc. whereas 2-ketogluconic acid mediated P solubilization
was found in Pseudomonas (Vyas and Gulati, 2009), B. rmus (Banik and
Dey, 1982) and R. leguminosarum (Halder et al., 1990). Organic P is
mineralized by acid phosphatases in Serratia sp. (Behera et al., 2017),
Pseudomonas (Gugi et al., 1991), Rhizobium (Abd-Alla, 1994), En-
terobacter, Citrobacter, Serratia and Klebsiella (Thaller et al., 1995), etc
whereas phytase activity has been shown in B. subtilis, P. putida, etc

Corresponding author.
E-mail address: shalini.rjk@nirmauni.ac.in (S. Rajkumar).

http://dx.doi.org/10.1016/j.micres.2017.05.005
Received 2 March 2017; Received in revised form 15 April 2017; Accepted 20 May 2017
Available online 30 May 2017
0944-5013/ 2017 Elsevier GmbH. All rights reserved.
B. Iyer et al.
(Richardson and Hadobas, 1997, Singh et al., 2014). Though P solubi-
lization has been studied in wide range of bacteria, succinate mediated
repression of P solubilization have been shown only in P. aeruginosa
(Patel et al., 2011) and K. pneumoniae (Rajput et al., 2013). Present
study is aimed at characterizing free living and symbiotic N2 xers for
inorganic P solubilization and its succinate mediated repression which
might be one of the reasons why these PSB which perform well in vitro
often fail in vivo.
2. Materials and methods
2.1. Isolation and characterization of isolates
Composite soil samples collected from elds of chharodi, district
Ahmedabad, Gujarat were clay loam soil having good water holding
capacity and pH of 7.4. Healthy chick pea plants, 2530 days after
germination were uprooted and packed into sterile polythene covers.
The plants were transported immediately to the laboratory where
healthy root nodules and rhizospheric soil gently adhering to the root
hairs were collected. Soil samples were serially diluted and plated onto
Luria Bertani Agar and incubated at 30 C for 2448 h. Root nodules
were surface sterilized with 0.1% HgCl2, crushed in sterile distilled
water, nodule sap was spread on Yeast extract mannitol agar with
congo red and incubated at 28 C for 4872 h. Colonies with distinct
morphology were plated onto Ashby's mannitol agar to check their
ability to grow on N free media. N2 xers were selected and char-
acterized biochemically according to Bergeys Manual of Systematic
Bacteriology. All selected isolates were characterized for lactose fer-
mentation, phenylalanine deaminase production, acetoin production,
citrate utilization, ability to hydrolyse starch, etc. Molecular identi-
cation was carried out through 16S rDNA sequencing using universal
primers F27 (5-AGA GTT TGA TCA TGG CTC AG-3) (Cheneby et al.,
2000) and R1492 (5-TAC GGT TAC CTT GTT ACG ACT T-3) (Wang
and Wang, 1996). Multiple sequence alignment was carried out with
sequence data available in the GenBank using BLASTn algorithm
(Altschul et al., 1997).
2.2. Characterization of isolates for P solubilization
To check the ability of isolates to solubilize TCP and RP, isolates
were spot inoculated on Pikovskayas agar (Pikovskaya, 1948) and
25 mM Tris buered rock phosphate (TRP) agar with methyl red pH
indicator (Gyaneshwar et al., 1998) respectively. 100 mM glucose was
added as sole carbon source. Clear halo around the colonies in Pi-
kovskayas agar plate and red coloration around the colonies in TRP
agar indicated TCP and RP solubilization respectively. P solubilization
was quantitatively estimated in Pikovskayas broth and 25 mM Tris
buered RP broth inoculated with test cultures and incubated at 30 C
for 4 days. Samples were withdrawn at 24 h intervals, centrifuged at
9000 rpm for 10 min and the cell free supernatant was used to de-
termine the pH change, residual glucose concentration and soluble P
using Ame's method (Ames, 1966).
2.3. HPLC analysis of organic acids produced
Isolates showing maximum TCP solubilization, E43 and RM1 were
grown in TRP broth with 100 mM Glucose as sole carbon source.
Cultures with minimum pH and maximum P release were selected for
HPLC analysis. Cells were pelleted down at 9000 rpm for 10 min at
25 C, supernatant was lter sterilized using 0.22-m Nylon lter and
subjected to HPLC analysis in Perkin Elmer series 200 with Hypersil
C18-column. pH of 25 mM KH2PO4 was set to 2.5 with orthophosphoric
acid and was used as a mobile phase at a ow rate of 1 mL/min.
Organic acids were detected using UV/VIS detector at 214 nm.
Gluconate, malate, citrate, acetate and succinate were taken as stan-
dards. Retention time of the organic acid and area under curve were
44
Microbiological Research 202 (2017) 4350
compared with that of standards to identify the type and concentration
of the organic acid produced.
2.4. Determining succinate mediated repression of P solubilization
To check succinate mediated repression of TCP and RP solubiliza-
tion, isolates were spot inoculated on Pikovskayas agar (Pikovskaya,
1948) and 25 mM tris buered RP agar (Gyaneshwar et al., 1998) with
50 mM glucose and 50 mM succinate. Repression of P solubilization
was quantitatively estimated in Pikovskayas broth and TRP broth with
carbon sources as indicated above, incubated at 30 C for 4 days.
Samples were withdrawn at 24 h intervals, centrifuged at 9000 rpm for
10 min and the cell free supernatant was used to determine the pH
decrease, residual glucose and soluble P using Ame's method (Ames,
1966).
2.5. Characterization of isolates for ammonia and IAA production
For checking ammonia production, all the isolates were grown in
peptone water at 30 C for 4872 h and NH3 production was tested
using Nessler's reagent (Cappuccino and Sherman, 1992). For checking
IAA production and related substances, isolates were inoculated in
Tryptophan broth and incubated at 30 C for 48 h. The concentration of
IAA produced was determined spectrophotometrically at 530 nm using
Salkowskis reagent (Gordon and Weber, 1951).
2.6. Characterization of isolates for enzyme production
2.6.1. Chitinase activity
Isolates were grown on Chitinase detection medium as described by
Kim et al. (2003). Chitinase assay was performed with colloidal chitin
as a substrate. Release of N-acetylglucosamine was estimated using
Dinitrosalicylic acid (DNS) (Miller, 1959). One unit of chitinase activity
was dened as the amount of enzyme that catalyzed the release of
1 mol of N-acetylglucosamine per mL per min.
2.6.2. Phytase activity
Phytase activity was determined in liquid phytate solubilization
media (PSM) (Engelen et al., 1994). One unit of phytase activity was
dened as the amount of enzyme that hydrolyzed 1 M of Ca-phytate
per ml per min.
2.6.3. Alkaline phosphatase activity
Alkaline phosphatase activity was checked as described (De Prada
et al., 1996). One unit of phosphatase activity was dened as the
amount of enzyme that hydrolyzed 1 M pNPP per mL per min.
2.7. Pot experiments
Fertile eld soil was collected and sterilized by repeated cycles of
autoclaving. Soil used for plant growth experiments was clay and loamy
with pH 7.5 and total P content of 0.10 g/L. All pots were lled with
about 1 kg sterile soil and watered with 150 mL autoclaved water.
Chick pea seeds were surface sterilized with 0.1% HgCl2 and allowed to
imbibe overnight. The seeds were bacterized overnight and then placed
at a depth of 5 cm in plastic pots with sterilized soil and watered with
sterile water. Pots were inoculated with the four isolates RS1, Pt1, E43
and PSB1 in all combinations which showed good P solubilization and
other PGP traits. Inoculated pots were maintained under greenhouse
conditions (10 h day and 14 h night cycle) at 25 C. Uninoculated plants
served as control. Plants were uprooted after 30 days of inoculation and
were scored for multiple plant growth parameters.
2.8. Statistical analysis
All the experiments were performed in triplicates and result values
B. Iyer et al.
are expressed as mean standard deviation. An analysis of variance
(ANOVA) was performed using the statistical program GraphPad Prism
Version 6.07. Statistical signicance was determined using the Holm-
Sidak method, with alpha = 5.000%.
3. Results
3.1. Isolation and characterization of isolates
About 50 morphologically distinct isolates from rhizospheric soil
sample and root nodules were selected for initial screening of N2 xa-
tion phenotype. A total of 31 isolates were identied to be N2 xers as
determined by their capability to grow on N free Ashby's mannitol agar
and were taken for further studies. Biochemical properties of all the
isolates are listed in Supplementary Table 1. Molecular characterization
showed that the isolates belonged to diverse genus like Enterobacter,
Acinetobacter, Erwinia, Pseudomonas, Rhizobium, Sinorhizobium, Ensifer,
Klebsiella, etc. Fig. 1 presents the maximum likelihood tree of all the
isolates along with their GeneBank accession numbers.
3.2. Characterization of isolates for P solubilization and its succinate
mediated repression
Out of the total 31 isolates characterized for P solubilization phe-
notype, 24 isolates could solubilize both TCP and RP. Maximum TCP
solubilization was found in Erwinia sp. E43 and Enterobacter sp. RM1
with P release of 850 g/mL and 812 g/mL and a minimum pH of 2.8
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Microbiological Research 202 (2017) 4350
Fig. 1. Maximum likelihood tree based on 16S rRNA
sequences of all the isolates along with their
GeneBank accession numbers.
and 2.9 respectively. However, when succinate was supplemented to
the medium along with glucose, 80% repression of P solubilization was
seen for both the isolates while repression in other isolates varied from
30 to 95%. Maximum of 95% repression of TCP solubilization was
found in Enterobacter cloacae Lh5. TCP solubilization in Pikovskaya's
broth, its succinate mediated repression and percent repression for all
the isolates is shown in Table 1. Maximum RP solubilization with P
release of 2087 g/mL and pH drop up to 3.3 was found for Erwinia sp.
PSB1, P release of 908 g/mL and pH 3.8 for Kosakonia cowanii Br5 and
529 g/mL P release and pH of 4.8 for Acinetobacter sp. SS1 was ob-
tained in 25 mM Tris buered RP medium. This was repressed by 87%,
42% and 38% respectively in the presence of 50 mM succinate while for
other isolates the repression in RP solubilization ranged between 38
and 100%. Complete repression of RP solubilization was found in Er-
winia sp. E43 and E. cloacae PSB8. P solubilization in all the isolates
were mediated by acid production manifested as red zone around the
colony on TRP agar plates as shown in Fig. 2. P release and pH change
in TRP broth with glucose, its succinate mediated repression and per-
cent repression for all the isolates is shown in Table 2.
3.3. HPLC analysis of organic acids produced
TRP broth samples with lowest pH were used for HPLC analysis.
HPLC prole of cell free supernatants of E43 and RM1 showed pro-
duction of 36 mM and 32 mM gluconic acid from 100 mM glucose
within two days of incubation as indicated by HPLC analysis. This
conrmed that acid production leading to reduction in pH was the main
B. Iyer et al. Microbiological Research 202 (2017) 4350

Table 1 Table 2
Succinate mediated repression of tricalcium phosphate solubilization in Pikovskaya's Succinate mediated repression of rock phosphate solubilization.
broth.
Isolate TRP broth with glucose TRP broth with glucose and % Decrease
Isolate Pikovskayas broth with Pikovskaya's broth with % Decrease succinate (*)
glucose glucose and succinate (*)
pH P (g/mL) pH P (g/mL)
pH P (g/mL) pH P (g/mL)
MB1 5.8 0.5 16.2 0.9 7.0 0.6 10.0 1 38
MB1 5.2 0.3 389 26 6.6 0.4 93 5 76 I91 5.8 0.4 142.8 11 7.3 0.5 6.9 0.8 95
I91 3.8 0.1 366 24 5.5 0.2 60 4 84 MB3 4.7 0.4 11.5 1 6.4 0.4 1.2 0.4 90
MB3 3.6 0.2 174 13 5.8 0.4 42 2 76 Br5 3.8 0.3 902.7 81 7.4 0.6 521.6 28 42
Br5 3.7 0.3 105 8 5.4 0.5 64 6 39 Ap15 2.6 0.2 168.6 13 4.1 0.3 133.6 11 21
Ap15 3.4 0.2 238 21 5.1 0.1 74 4 69 RM1 7.6 0.6 193.4 16 8.1 0.7 44.4 4 77
RM1 2.9 0.1 812 56 4.4 0.2 154 9 81 SM1 2.6 0.1 49.5 4 4.0 0.3 8.3 0.7 83
SM1 4.1 0.5 189 14 5.3 0.6 40 2 79 PSB1 3.3 0.2 2087.4 89 4.8 0.3 369.1 28 87
PSB1 3.5 0.2 403 25 5.1 0.1 95 4 76 PSB4 2.7 0.2 46.8 3 8.3 0.7 11.1 1.1 76
PSB4 3.3 0.4 562 27 7.2 0.4 95 3 83 PSB8 3.7 0.3 55.2 4 6.1 0.4 0.7 0.1 99
PSB8 3.0 0.1 397 34 5.4 0.5 44 5 89 PSB12 4.1 0.2 34.6 3 7.4 0.6 3.1 0.2 91
PSB12 4.5 0.2 362 38 5.7 0.3 30 2 92 Lh4 3.0 0.2 170 8 4.5 0.3 14 0.3 92
Lh4 3.7 0.2 438 21 6.4 0.1 124 9 72 Lh5 2.7 0.3 38 2 4 0.3 10 1.1 74
Lh5 4.4 0.5 118 12 7.1 0.2 6 1 95 Gh1 2.8 0.2 54 4 4.1 0.3 8.2 0.2 85
Gh1 4.5 0.1 343 31 7.3 0.5 82 3 76 Wt6 6.1 0.4 16.7 2 6.3 0.4 8.1 0.7 51
Wt6 5.1 0.4 165 15 6.7 0.4 38 4 77 Pt1 2.6 0.3 144 9 3.9 0.2 96 6.8 93
Pt1 3.3 0.1 238 9 5.2 0.2 121 7 32 E43 5.6 0.4 36 2 6.8 0.5 0 0 100
E43 2.8 0.2 850 58 4.4 0.1 166 11 80 AC1 6.5 0.3 12 2 7.2 0.6 2 0.4 83
AC1 3.0 0.1 178 12 5.5 0.2 128 9 28 RS1 2.9 0.2 57 4 4.3 0.3 22 1.6 61
RS1 3.3 0.4 256 21 6.8 0.3 35 2 86 RS2 7.3 0.5 120 9 8.2 0.7 50 3.7 58
RS2 3.8 0.3 195 13 5.2 0.5 12 1 94 RS3 3 0.3 133 8 4.5 0.3 16 1.1 88
RS3 4.3 0.4 246 18 7.2 0.4 35 2 86 RS4 6.7 0.5 14 1 8.4 0.7 3.5 0.8 75
RS4 3.8 0.2 322 24 6.8 0.6 156 8 52 SS1 4.8 0.4 529 45 7.9 0.6 330 24 38
SS1 3.4 0.2 134 11 5.1 0.4 51 4 62 SS2 3.9 0.3 70 6 5.5 0.4 7.1 0.7 90
SS2 4.4 0.4 486 38 6.5 0.2 89 6 82
Values are mean standard deviation of 3 independent observations.
Values are mean standard deviation of 3 independent observations. * Signicant as compared to cells grown in glucose as sole carbon source (P 0.05).
* Signicant as compared to cells grown in glucose as sole carbon source (P 0.05).
Table 3. All the isolates produced ammonia as indicated by brown
mechanism of P release. colouration of the medium upon addition of Nessler's reagent. All iso-
lates were also positive for production of IAA and related substances as
3.4. Characterization of other PGP properties indicated by pink colouration of the cell free supernatant upon addition
of Salkowski's reagent. Only few isolates had chitinase activity with the
The results of PGP properties of all the isolates is described in

Fig. 2. RP solubilization by the isolates in TRP agar with glucose and glucose + succinate. (For interpretation of the references to colour in this gure legend, the reader is referred to the
web version of this article.)
Figure shows rock phosphate solubilization by the isolates in 25 mM tris buered RP agar with 100 mM glucose (a) and repression of P solubilization by the isolates in 25 mM tris buered
agar with 50 mM glucose + 50 mM succinate. Development of pink zone in (a) indicates RP solubilization mediated by organic acid production whereas lighe color change of the media
to pink in (b) shown partial to complete repression of P solubilization.

46
B. Iyer et al. Microbiological Research 202 (2017) 4350

Table 3 Group of benecial bacteria widely known as PGPR are known to co-
Plant growth promoting traits of all isolates. lonize plant roots thereby improving plant growth by multiple me-
chanisms, one of which is P solubilization. P plays an important role in
Isolate IAA Chitinase activity Phytase activity Alkaline
production (*) phosphatase virtually all plant process, but its availability is limited in soil because
activity of which studies about microbial P solubilization have gained im-
portance. In recent years, multiple studies have characterized bacteria
MB1 67 3.1 0.99 0.1* 1.40 0.2* 6.7 0.5*
for ecient P solubilization. This study was aimed at isolating N xing
I91 70 4.1 0.0 11.13 0.1* 7.2 0.9*
MB3 20 0.5 44.8 3.6* 4.10 0.3* 0.7 0.0* bacteria and studying their multiple PGP traits as they are important for
Br5 13 0.2 0.0 0.53 0.1 5.3 0.4* plant N nutrition (Turk et al., 2011).
Ap15 162 7.4 1.14 0.33* 0.0 3.6 0.3* Isolates belonging to multiple bacterial classes were obtained from
RM1 173 9.7 0.0 2.43 0.5* 2.2 0.1* chick pea root nodules and rhizospheric soil. All the isolates were
SM1 115 5.3 0.0 0.90 0.0* 9.4 0.8*
evaluated for multiple PGP traits with special emphasis on P solubili-
PSB1 37 1.6 0.0 2.07 0.1* 1.4 0.2*
PSB4 27 2.1 0.91 0.12* 1.47 0.2* 0.5 0.0* zation and the eect of succinate on P solubilization. In this study, most
PSB8 25 1.1 0.0 0.0 4.1 0.3* of the isolates screened could solubilize both TCP and RP except Bacillus
PSB12 30 1.2 0.0 0.30 0.0 1.0 0.1* sp. RX1, Klebsiella pneumoniae Lh3 isolated from chick pea rhizospheric
Lh3 246 18.3 1.90 0.2* 1.43 0.1* 0.1 0.0
soil and the strains belonging to Rhizobiaceae isolated from the root
Lh4 123 8.5 0.0 0.20 0.0 1.6 0.2*
Lh5 116 7.3 0.0 1.00 0.1* 2.7 0.2* nodules. Results indicated that most members of Enterobacteriaceae fa-
Gh1 97 6.2 0.0 0.0 3.1 0.2* mily were ecient P solubilizers. The results of TCP solubilization in
Td22 140 9.3 0.0 0.0 4.9 0.3* Pikovskaya's broth was higher than those reported as 110130 g/mL
Wt6 26 1.3 0.46 0.08* 2.10 0.0* 4.8 0.4* for Klebsiella sp. (Rajput et al., 2013), 35 g/mL for B. cepacia and
Pt1 175 11.3 6.52 0.86* 0.0 0.6 0.1*
51 g/mL for Pseudomonas sp. (Rodriguez and Fraga, 1999). RP solu-
E43 141 12.1 0.0 0.0 2.5 0.2*
AC1 21 0.4 0.15 0.03 0.73 0.1* 0.8 0.1* bilization was also higher that the earlier reports where P release of
RS1 167 10.2 0.0 1.13 0.1* 0.7 0.0* 1312 g/mL by P. uorescens (Oteino et al., 2015), 63 g/mL and
RS2 28 1.5 0.0 0.80 0.0* 0.6 0.0* 76 g/mL by P. aeruginosa (Patel et al., 2011) have been shown. 50 g/
RS3 21 0.8 0.0 0.0 1.2 0.1*
mL and 77 g/mL P release from RP has been shown by Citrobacter sp.
RS4 29 1.5 0.0 0.10 0.0 6.1 0.6*
SS1 29 1.1 0.0 0.07 0.0 1.1 0.1*
(Gyaneshwar et al., 1999; Patel et al., 2008) in RP medium under
SS2 88 4.2 0.0 0.73 0.0* 2.7 0.4* 100 mM Tris buering conditions. These ecient P solubilizers char-
RN1 81 4.2 0.0 0.0 0.2 0.0 acterized in this study may act as better candidate for PGP. However, all
CN1 32 1.7 3.11 0.3* 0.0 0.0 the P solubilizers tested showed 30100% repression of P solubilization
UN1 126 9.9 2.05 0.2* 1.73 1.0* 0.1 0.0
when media with glucose was supplemented with succinate. Succinate
RN2 39 2.1 0.18 0.0 0.13 0.01 2.4 0.4*
mediated repression of P solubilization has been reported for P. aeru-
* Signicant as compared to uninoculated controls (P 0.05). IAA production is in- ginosa (Patel et al., 2011) and K. pneumoniae (Rajput et al., 2013) which
dicated in g/mL and enzyme activities are shown as uM/min/mg protein. Values are solubilized P through release of gluconic acid and oxalic acid, respec-
Mean Standard deviation of 3 independent observations. tively. A scheme outlining the eect of CCR on P solubilization is shown
in Fig. 4. Isolates E43 and RM1 produced gluconic acid as the major
maximum being 44.8 U/mL for Enterobacter sacchari MB3. Maximum acid responsible for P solubilization. Gluconic acid produced in gram
phytase activity of 11.3 U/mL was obtained with Enterobacter sp. I91. negative bacteria through periplasmic glucose oxidation pathway by
The isolates showed negligible alkaline phosphatase activity with quinoprotein Gcd is known to result in most ecient P solubilization
maximum activity of 9.4 U/mL with SM1 and 6.1 U/mL for Acineto- (Hilda and Fraga, 1999). Gluconic acid mediated P solubilization have
bacter sp. RS4. been shown in Rahnella aquatilis, Erwinia herbicola, Pseudomonas cepacia
and Enterobacter asburiae (Patel et al., 2011).
3.5. Pot experiments Rhizosphere is rich in amino acids, organic acids, sugars, vitamins
and signals that attract microbes which are capable of metabolizing
Fig. 3 represents the PGP traits of all the isolates tested for plant plant exudates, enabling them to proliferate in the rhizosphere (Bais
growth in pot experiments. Potted plants inoculated with appropriate et al., 2006). But the presence of C4 acids like succinate in the rhizo-
cultures were grown for 30 days after inoculation. The uprooted plants spheric root exudates may cause repression of P solubilization which
were washed well and scored for multiple plant growth promotion could be a reason for the failure of PSBs in vivo even though they per-
parameters like root length, shoot length, root: shoot ratio (length), root form well in vitro. This repression may occur as a consequence of a
weight, root dry mass, shoot weight and shoot dry mass. Results ob- mechanism termed as Carbon catabolite repression (CCR) which de-
tained for the pot experiments with Enterobacter sp. RS1, Enterobacter termines the hierarchy of carbon source utilization in bacteria (Gorke
hormaechei Pt1, Erwinia sp. E43 and Erwinia sp. PSB1 are shown in and Stulke, 2008) and also inuences the bacterial virulence, con-
Table 4. RS1 and Pt1 + RS1 showed maximum root length while RS1 tributes to resistance to various antibiotics and chemicals, etc. Me-
and E43 + Pt1 showed maximum shoot length. Maximum of 1.8 and chanism of CCR prevails in most of the metabolically versatile hetero-
1.7 fold increase in root-shoot ratio of chick pea plants was obtained trophic bacteria for selectively assimilating preferred carbon source
with E43 + PSB1 and Pt1 + RS1 inoculation respectively. 2.2 and 2.1 from a mixture of potential carbon sources (Rojo, 2010). 510% of the
fold increased root weight was obtained for E43 + Pt1 + RS1 and bacterial genes are subjected to CCR as it is one of the most important
Pt1 + RS1 inoculation while 1.5 fold increase in shoot weight was regulatory phenomena (Moreno et al., 2001). This mechanism should
obtained with RS1 and E43 respectively. 2.5 and 2 fold increase in root be considered while evaluating in vivo ecacy of any PGPR for any
dry mass was obtained with Pt1 + RS1 and E43 inoculation while 2.4 plant growth promoting trait as this mechanism may critically inuence
and 1.8 fold increase in shoot dry mass was obtained with their eld performance.
E43 + Pt1 + RS1 and E43 + PSB1 inoculation, respectively. All the isolates were positive for ammonia production which is an
indirect PGPR trait promoting plant growth. Ammonia production re-
4. Discussion duces ethylene levels in plant (Glick et al., 1998). Almost 80% of the
rhizospheric microorganisms can produce IAA as a secondary metabo-
Plant rhizosphere is rich in nutrients and hence inhabits multitude lite (Patten and Glick, 1996) which is critical for plant growth, devel-
of microorganisms which inuence plant growth in numerous ways. opment and defence responses (Santner et al., 2009). Amino acid

47
B. Iyer et al. Microbiological Research 202 (2017) 4350

Fig. 3. PGP traits of isolates used for pot experiments.

(a) Tricalcium phosphate and rock phosphate solubilization by E43, PSB1, Pt1 and Rs1 (b) IAA production (c) Chitinase, phytase and alkaline phosphatase activities. Values are
mean standard deviation of 3 independent observations

Table 4
Eect of bacterial inoculation on chick pea growth.

Isolate Root length Shoot length (cm) Root weight Shoot weight (mg) Root dry mass (mg) Shoot dry mass
(cm) (mg) (mg)

Control 14.7 1.0 25.3 0.6 63.3 5.8 433 21 46 7.0 210 25.0
E43 18.0 1.5* 22.0 1.5* 113 25.4* 633 34* 90 7.0* 280 24.0*
PSB1 21.2 1.3* 23.7 1.2 83.6 5.8* 526 42 70 5.0* 240 20.0*
Pt1 19.3 1.5* 25.0 1.0 66.7 5.3 533 24 60 4.5* 250 18.0*
RS1 20.5 0.9* 28.0 + 1.6* 96.7 5.8* 643 54* 70 4.7* 220 17.8
E43 + PSB1 19.0 3.5* 19.2 1.0* 90 10.0* 556 38 80 7.7* 300 28.0*
E43 + Pt1 19.5 2.3* 25.9 0.6 96.7 7.6* 746 58* 60 5.7* 220 15.7
E43 + RS1 14.8 1.5 24.7 1.8 66.7 3.8 530 34 50 4.7 240 14.6*
PSB1 + Pt1 18.1 0.6* 22.7 2.5 113 12* 516 35 50 3.0 220 13.4
PSB1 + RS1 15.0 1.8 26.0 1.7 90 8.5* 596 38* 50 4.1 250 18.0*
Pt1 + RS1 20.1 1.0* 20.8 1.7* 130 15* 616 54* 114 11.8* 370 27.0*
E43 + PSB1 + Pt1 14.8 0.3 18.5 1.8* 50 6.8 500 35 35 2.8 260 17.2*
E43 + PSB1 + RS1 14.3 1.4 24.5 2.6 70 7.0 566 54 64 5.4* 265 18.9*
E43 + Pt1 + RS1 16.2 1.8 21.7 0.6* 140 14* 586 48* 86 6.7* 500 37.9*
PSB1 + Pt1 + RS1 13.5 0.5 24.3 1.6 90 8.0* 596 57* 50 3.7 250 16.4*
E43 + PSB1 + Pt1 + RS1 16.9 1.6 21.8 0.8* 63 4.8 510 42 40 3.7 200 13.9

Values are Mean Standard deviation of 9 plants.

Total amount of cells added to the plants were kept constant.
* Signicant as compared to uninoculated control pots (P 0.05).

48
B. Iyer et al.
Fig. 4. Eect of CCR on P solubilization.
(a) Soil P is in a complex form unavailable for direct plant uptake and hence limits plant growth and productivity (b) Phosphate solubilizing bacteria harbouring the soil can convert this
complex unavailable phosphates to simple available forms through multiple mechanisms including organic acid secretion, which may increase plant growth and productivity (c)
However, root exudates secreted by plants have C4 acids like succinate which by the mechanism of CCR may inhibit P solubilization, again limiting the plant growth and productivity.
tryptophan is an important precursor of IAA synthesis and the con-
centration of IAA produced increases with increase in tryptophan
concentration (Zaidi et al., 2009). The concentration of IAA produced
was higher by the test strains compared to earlier reports i.e. 19 g/mL
of IAA production by K. pneumoniae (Rajput et al., 2013), 140 g/mL by
Acinetobacter sp. and 107 g/mL by Enterobacter sp. (Kumar et al.,
2012). Chitin, the second most abundant natural polymer forms com-
ponent of fungal cell wall which can be degraded by exochitinases and
endochitinases (Dahiya et al., 2006). Aeromonas, Bacillus, Serratia,
Streptomyces and Vibrio are the bacterial genera best known for chit-
inase production (Cody, 1989). Maximum chitinase activity obtained
was higher compared to 15.4 U/mL by S. marcescens (Xia et al., 2011)
and 31.6 U/mL by Streptomyces sp. (Thiagarajan et al., 2011). Previous
reports suggest phytase activity in Pseudomonas sp. (Richardson and
Hadobas, 1997), Citrobacter sp. (Kim et al., 2003), etc. Phytase activity
was maximum for Enterobacter sp. Presence of acid phosphatase has
been shown in Enterobacter sp., Citrobacter sp. (Thaller et al., 1995),
Pseudomonas sp. (Gugi et al., 1991), etc. However, studies regarding
alkaline phosphatase activities in bacteria have been limited.
Pot experiments with chick pea indicated that E43, Pt1 + RS1 and
E43 + Pt1 + RS1 inoculations improved almost all the plant growth
parameters considered which might be because all of them solubilized P
and produced signicant concentrations of IAA and NH3. Response of
plants to dierent isolates was variable which may be because of their
individual traits and rhizospheric competencies (Majeed et al., 2015).
Increase in root number, length of lateral roots and root hair elongation
leads to increased uptake of water and minerals, thereby increasing the
growth of whole plant (Dobbelaere et al., 1999). PGPR inoculation also
leads to increased root and shoot biomass (Walker et al., 2012a,b). As P
solubilization may get repressed under soil conditions, chick pea plant
growth promotion by these PSBs may be attributed to other PGP traits
as their inoculation lead to a signicant increase in the plant growth
compared to the uninoculated controls. If the repression of P
49
Microbiological Research 202 (2017) 4350
solubilization by the components of root exudates is relieved by gen-
erating mutants with relieved CCR, mutant inoculation may further
increase plant growth compared to wild types thereby performing
better under soil conditions. Our previous report on succinate mediated
repression of oxalic acid mediated P solubilization in two Klebsiella
pneumoniae strains showed iclR, repressor of aceBAK operon to be the
key regulator of repression. Consequently, generation of iclR knockout
mutants in both the strains relieved repression of P solubilization with
54% and 59% increase in soluble P release and improved plant growth
promotion traits compared to their respective wild types even in pre-
sence of succinate (Rajput et al., 2015).
Taken together, most of the isolates tested had ability to solubilize
inorganic P and mineralize organic P in addition to other PGP traits. All
the P solubilizers were susceptible to succinate mediated repression of P
solubilization. Pot experiments showed signicant improvement of
chick pea plant growth by PGPR inoculation which shows that organ-
isms with multiple PGP traits can inuence plant growth in multiple
ways. Such types of studies are important for better exploiting poten-
tials of PGPR under eld conditions and for their use as potential mi-
crobial inoculants.
Funding
This work was supported by Gujarat State Biotechnology Mission
(GSBTM), Department of Science and Technology, Government of
Gujarat (Grant number: GSBTM/MD/PROJECTS/SSA/1408/2014-15).
Conict of interest
The authors declare that they have no conict of interest.
B. Iyer et al.
Acknowledgements
The authors would like to acknowledge Gujarat State Biotechnology
Mission (GSBTM), Department of Science and Technology, Government
of Gujarat for the nancial grant (Project sanction number: GSBTM/
MD/PROJECTS/SSA/1408/2014-15), Department of Science and
Technology (DST), Government of India for providing INSPIRE
Fellowship to Bhagya Iyer (IF130895) and Council of Scientic and
Industrial Research (CSIR), Government of India for providing Senior
Research Fellowship to Mahendrapal Singh Rajput (Ack No. 113827/
2K12/1). Authors would also like to acknowledge Nirma Education and
Research Foundation (NERF), Nirma University for providing basic
infrastructure in supporting this research work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.micres.2017.05.005.
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