Acta Agriculturae Scandinavica, Section B — Soil & Plant

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PGPRs of plum (Prunus domestica) rhizosphere

enhance plant growth and antagonise fungal
activity in vitro

Izhar Ali, Tariq Sultan, Fazli Subhan, Kashif Syed Haleem, Nighat Sultana &
Isfahan Tauseef

To cite this article: Izhar Ali, Tariq Sultan, Fazli Subhan, Kashif Syed Haleem, Nighat Sultana &
Isfahan Tauseef (2018) PGPRs of plum (Prunus domestica) rhizosphere enhance plant growth
and antagonise fungal activity in vitro, Acta Agriculturae Scandinavica, Section B — Soil & Plant
Science, 68:4, 367-378, DOI: 10.1080/09064710.2017.1410565

To link to this article: https://doi.org/10.1080/09064710.2017.1410565

Published online: 06 Dec 2017.

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ACTA AGRICULTURAE SCANDINAVICA, SECTION B — SOIL & PLANT SCIENCE, 2018
VOL. 68, NO. 4, 367–378
https://doi.org/10.1080/09064710.2017.1410565

PGPRs of plum (Prunus domestica) rhizosphere enhance plant growth and

antagonise fungal activity in vitro
Izhar Alia, Tariq Sultanb, Fazli Subhana, Kashif Syed Haleema, Nighat Sultanac and Isfahan Tauseefa
a
Department of Microbiology, Hazara University, Mansehra, Pakistan; bLand Resource Research Institute, NARC, Islamabad, Pakistan;
c
Department of Biochemistry, Hazara University, Mansehra, Pakistan

ABSTRACT ARTICLE HISTORY

Plant growth promoting rhizobacteria (PGPR) promote the plant growth by various direct and Received 17 September 2017
indirect mechanisms. The present study was undertaken to isolate and characterise the PGPRs of Accepted 23 November 2017
plum (Prunus domestica) rhizosphere in Pakistan. A total of 95 rhizobacteria were isolated, out of
KEYWORDS
which 40 strains were selected on the basis of morphological, biochemical and Gram staining Bio-fertilizer; diversity; PGPR;
characteristics. The selected isolates were screened for in vitro plant growth promoting potential plant growth; plum
and were subsequently evaluated for host plant growth promotion. The selected isolates
demonstrated strong lytic enzymatic activities and were able to produce ammonia, siderophore,
Hydrogen cyanide along with capability of phosphate solubilisation. Moreover, the results
showed a significant growth suppression of pathogenic Fusarium oxysporum and Rhizoctonia
solani in an in vitro assay. The plant microbe interaction study was carried out using 11 most
efficient rhizobacterial strains inoculated to roots of plum plants. The inoculated PGPRs
significantly augmented the leaves number per shoot, shoot diameter, shoot length and plant
height. The inoculation also significantly increased the chlorophyll contents of leaves,
concentration of micro and macro nutrients compared with control. The current study shows the
importance of these selected PGPRs as bio-fertilizer to improve the health and productivity of
plum species in Pakistan.

Introduction
Despite considerable development in irrigation and
other technologies, the demand of fruits and vegetables
is a challenge for our agriculture sector so as to feed the
fast growing world population. As many Asian countries
hugely rely on agriculture for their economic stabilis-
ation, an enhancement in agricultural productivity is
required to meet the increasing demand of food by
rapidly growing population. Unfortunately, most of our
agriculture production is dependent on use of chemical
fertilisers which have not only disturbed the environ-
mental ecology but also posing a considerable challenge
to human health (Iavicoli et al. 2017).
One of the recent biotechnological approaches,
appreciated and widely accepted by both agronomists
and environmentalists, is the use of PGPRs as biofertili-
zers which are not only considered safe but also
provide a good economical alternative to chemical ferti-
lisers (PS and SB 2016; Alori et al. 2017; Mahanty et al.
2017). Plant Growth-Promoting Rhizobacteria are free-
living soil bacteria that aggressively colonise the rhizo-
sphere/plant roots. Once applied to seed or crop roots,
the PGPRs enhance the growth and yield of plants
through mobilising nutrients such as ammonia and
phosphate in soil (Islam et al. 2015), producing numerous
plant growth regulators and protecting from phyto-
pathogens or insects (Compant et al. 2010; Jin et al.
2017; López-Ráez et al. 2017), bioremediating the toxic
heavy metals (Ma et al. 2011; Sobariu et al. 2016) and pes-
ticide degradation/tolerance (Ahemad and Khan 2012a,
2012b). In particular, Pseudomonas, Stenotrophomonas,
and Bacillus have been successfully used in attempts to
control plant pathogens and increase plant growth
(Bais et al. 2004; Idris et al. 2007).
The discovery of new native bacterial strains identifi-
cation, characterisation and application in the targeted
rhizosphere of specific crops are more selective for
crop production improvement (Keating et al. 1995; Chah-
boune et al. 2011). Due to the need of biofertilizer, analy-
sis of plants and region specific microbes would be more
efficient tool for improving the production of biofertilizer
(Deepa et al. 2010).
Plum is widely grown as an ornamental or fruit tree
that, depending upon geographical area, blossoms in

CONTACT Isfahan Tauseef isfahan@hu.edu.pk Department of Microbiology, Hazara University, Mansehra, Pakistan
Supplemental data for this article can be accessed https://doi.org/10.1080/09064710.2017.1410565.
© 2017 Informa UK Limited, trading as Taylor & Francis Group
368 I. ALI ET AL.
different months of a calendar year. Approximately 40
different species of plum with an average height of 5–8
metres have been reported so far. The plum fruit is
either ingested directly or can be used for making
other products such as juices, plum wine, pickles jams
and prunes. In Pakistan, the plum is mostly grown in
the Northern region where it blossoms in early spring
and fruit is marketed in May. The main soil borne patho-
gen that affect the plants worldwide include the Fusar-
ium oxysporum and Rhizoctonia solani. (Lee et al. 2017;
Lyu et al. 2017). Keeping in view the role of PGPRs in
the growth, yield and fruit quality enhancement of
other fruits such as sour cherry (Prunus cerasus L.),
sweet cherry (Prunus avium L) and apricot (Prunus arme-
niaca L. cv. Hacihaliloglu) reported through various
studies (Esitken et al. 2003; Akca and Ercisli 2010;
Arikan and Pirlak 2016), it is not out of place to expect
an increased yield of plum through discovery and appli-
cation of selected PGPRs capable of restricting the
growth of soil borne fungal pathogens and able to
enhance the nutrients uptake.
As no data is available regarding the rhizosphere
microbiome of plum native to Swat valley area, this
study was conducted to isolate and study the diversity
of PGPR from the rhizosphere of plum. The identification
and characterisation of PGPR populations indigenous to
plums rhizospheres is critical to discover the strains that
can be utilised to improve the growth and pathogenic
suppression in plums. To the best of our knowledge,
this is the first report which describes the isolation and
positive effect of PGPR application on plum grown in
Pakistan.
Materials and methods
The study site and sample collection
To isolate and specify the PGPRs for their plant growth
characteristics and antagonistic activity, experimentation
was carried out in the Laboratory of Soil Biology and Bio-
chemistry, Land Resources Research Institute (LRRI),
National Agricultural Research Centre (NARC), Islamabad.
The study area (District Swat, KPK) lies in North West of
Pakistan with sub-humid temperate conditions, slightly
sloppy land and stretches of valleys where stone fruits
are conventionally cultivated. Among all fruits, plum
(Prunus domestica) is the major fruit in this locality.
Hence, considering the suitability of this area for plum
production, soil samples were collected from the root
system of plum plants grown at study area. As the
PGPR microbial community resides intact or close to
root soil system, the samples were collected from 0 to
20 cm depth at random from 10 different plants and
mixed well. The field-moist soil was filtered through a
4 mm sieve to eliminate coarse rock and plant material
and immediately stored at 4°C.
Isolation of plant growth promoting rhizobacteria
(PGPR)
The Luria Agar (LA) media were used for isolation of
different PGPR. The PGPRs were isolated from the rhizo-
sphere, rhizoplane and endorhizosphere of the plum
plant by serial dilution technique described previously
(Johnson and Curl 1972). The prepared serial dilutions
from each sample were thoroughly spread on Luria
Agar plates and incubated at 28°C for 24 h. The isolates
were purified by re-streaking on fresh media plates and
their morphological characteristics such as colour, form,
shape, elevation, margins and opacity were recorded.
Biochemical characterisation of isolates
Gram’s staining
Bacterial Gram’s staining was done according to the
method described by Vincent and Humphrey (1970).
Crystal violet, iodine solution and safranin were used
according to the standard protocol and smears were
studied microscopically at 100X lens under bright field
microscope.
Ammonia production
Cappuccino and Sherman (1992) method was used to
test the bacterial isolates for Ammonia production. Cul-
tures were grown in tubes with peptone water and incu-
bated for 4 days. One ml of Nessler’s reagent was added
in each tube after incubation. Yellow to brownish colour
was recorded positive for production of ammonia.
Siderophores production
Bacterial strains were evaluated for Siderophores pro-
duction on CAS (Chrome azorole s agar) agar and the
method described by Schwyn and Neilands (1987).
Blue colour L.B agar plates containing dye chrome
azurole S were prepared and a loop full fresh culture
was spotted on CAS agar plates. The plates were incu-
bated for 5 days at 30°C. Formation of orange halo
zone around the bacterial colony was recorded as posi-
tive for production of Siderophores.
Hydrogen cyanide
To evaluate the cultures for Hydrogen cyanide pro-
duction Lorck (1948) method was used. Bacterial isolates
on nutrient agar medium were mixed with glycine (4/4 1-
gl). The production of cyanide was detected after four
days of incubation, using picrate/Na2Co3 paper fixed to
 ACTA AGRICULTURAE SCANDINAVICA, SECTION B — SOIL & PLANT SCIENCE
the underneath of the Petri-dish lids which were
wrapped with parafilm, Colour of filter paper changed
from yellow to orange, red, brown, or reddish brown
indicated weak, moderate, or strong cyanogenic poten-
tial, respectively.
Motility
Motility of the selected isolates was determined by
Barrow and Feltham (1993) method. Motility test
medium was prepared in tubes which were inoculated
with isolate by means of straight wire loop up-to a
depth of 5 mm. The inoculated tubes were incubated
for 24 h and bacteria which moved from stab line were
noted as motile while growth in straight line were
recorded as non-motile.
Indole acetic acid (IAA) production and
quantification
Indole acetic acid (IAA) production was detected as
described by Brick et al. (1991). Cultures were injected
in tryptone broth in tubes and incubated at 35°C (+/2°
C) for 24–48 h. To test for indole synthesis, five drops
of Kovács reagent were directly added to the tube. An
instant formation of cherry red ring at the top of
medium indicated the production of auxins. Quantifi-
cation of IAA was determined by the method used by
Asghar et al. (2002). Quantification of IAA was made in
µg/ml by determining the regression equation of stan-
dard curve.
Phosphate solubilisation
All bacterial cultures were tested on Pikoviskaya’s agar
medium by using method of Nautiyal (1999). Bacterial
strains were streaked on petri plates having Pikoviskaya
agar medium by using tooth picks. Formation of halo
zone around bacterial strains after 7 days incubation
period at 28°C was observed which indicated solubil-
isation of insoluble phosphate. The ability of the bacteria
to solubilise insoluble phosphate was pronounced by the
solubilisation index by Edi-Premono et al. (1996) method
as
Solubilization index (SI) = Colony diameter
halo zone diameter
+ To observe the effect of PGPRs on growth of plum (1-year-
colony diameter
Enzyme production characterisation
Bacterial strains were examined for their catalase activity
according to MacFaddin (2000). Freshly grown bacterial
culture was transferred on glass slide and mixed with
appropriate quantity of 3% hydrogen peroxide (H2O2).
369
Formation of oxygen bubbles within 10 s confirmed
the strain as catalase positive. Oxidase test was done as
described by Barrow and Feltham (1993). In oxidase
reagent solution, a small piece of filter paper was
soaked and a wire loop full of bacteria was rubbed on
the filter paper. Production of blue colour within 10 s
indicated positive result. The amylase production was
determined as described by Ashwini et al. (2011). The
culture grown on starch medium was drowned with
iodine solution to observe the formation of blue colour
which faded rapidly.
To check the protease production, bacterial isolates
were streaked on skimmed milk agar medium and incu-
bated at 35°C for 48 h (Kazempour 2004). Formation of
halo zone around bacterial colony was taken as positive
result. Finally, the production of pectinase by bacterial
cultures was detected on specific medium. After 24 h
of incubation, culture was flooded with diluted HCL sol-
ution. Formation of clear zone was the positive result of
pectinase production (Namasivayam et al. 2011).
Dual culture assay
To determine the antagonistic effect of bacteria, method
defined by Sivan et al. (1987) was used. The bio-control/
antagonistic activity of PGPR isolates was performed
against two soil-borne fungal pathogens which include
Fusarium oxysporum and Rhizoctonia solani. Five mm
fungal mycelial discs of each pathogen were positioned
at the one edge of the petri plate containing Potato dex-
trose agar (PDA) medium. Apposite to fungal disc at
other edge, bacterial culture was streaked and incubated
for 5 days at 28°C. Petri plate spotted with fungal disc
only served as a control. Comparison was made among
the mycelial growth of the pathogen and that of
control. The experiment was performed in triplicates
for each treatment. The percentage growth inhibition
of the fungal pathogen was calculated using formula:
Percentage Growth Inhibition (PGI)
= [1 − (Fungal growth/control growth)] × 100
Greenhouse experiment to evaluate the effect of
PGPRs on young plum plants
old), an in vivo study was performed on 1-year-old plum
plants in a controlled environment using complete ran-
domise design method. The green house was used to
reduce the effects of external factors i.e. insects and
extremes of temperature. The treatments were done in
3 replicates while a control in triplicate with no inoculation
was also set up (Table 1). Two treatments with mixed
 370
I. ALI ET AL.
Table 1. Summary of all tests for the selected 11 PGPRs strains.
Strain’s Indole HCN Siderophore Phosphate Ammonia Amylase Pectinase Protease Oxidase Catalase Motility Antagonistic Activity Antagonistic Activity
Code test test Test solubilisation Test Test test Test Test Test Test (R. solani) (F. oxysporum)
IPL-02 − ++ − ++ + + ++ ++ + + − + +
IPL-08 − − − + + + ++ ++ + + + +++ +++
IPL-09 − − ++ ++ +++ +++ +++ + − + − +++ +
IPL-10 − − − ++ +++ +++ +++ +++ − + + − ++
IPL-13 − − ++ + +++ ++ + +++ + + + +++ +++
IPL-17 + − − + ++ +++ ++ ++ − + + +++ +
IPL-18 − − − +++ ++ + ++ +++ − + − − +
IPL-19 ++ − − +++ ++ ++ ++ +++ + + + + −
IPL-24 − − − + ++ + +++ ++ − + + − +
IPL-27 − +++ − ++ + + + + + + + ++ ++
IPL-29 − − − + +++ +++ ++ ++ − + − +++ +++
For Biochemical parameters: +shows weak positive, ++ shows moderate positive, +++ represent strong positive, while – shows negative result for production.
For Phosphate Solubilisation:
For hydrolytic enzymes Activity: +shows Low Enzyme Activity, ++ shows Moderate Enzyme Activity, +++ shows strong enzyme activity.
For Antifungal activity: +shows weak antagonistic Activity, ++ shows moderate antagonistic Activity, +++ represent strong antagonistic Activity, while – represents NO Activity.
The table shows the summary of all testes for the selected 11 PGPRs strains. The +shows weak results, ++ shows moderate results, +++ represent strong results, while −shows negative result.
 ACTA AGRICULTURAE SCANDINAVICA, SECTION B — SOIL & PLANT SCIENCE
isolates designated as MIX-1 (IPL-9, 13, 17, 19, 27, 29) and
MIX-2 (IPL-02, 08, 10, 18, 24) were also performed. Both
mix groups were generated by random mixing of above
described 11 selected/ positive PGPR strains.
Briefly, the young plants (1-year-old) were collected
from Swat region and their roots were washed with tap
water. The roots were sterilised with Clorox for 1 min fol-
lowed by their washing (2–3 times) with double distilled
water. For every bacterial treatment (in triplicate), slurry
was made and the roots of each plant was inoculated
with testing strain followed by planting in a sterilised
and labelled pot containing 10 kg of previously auto-
claved soil. The soil used was moderately to highly cal-
careous with a pH range of 7.8–7.9. During growth
experiment, natural ventilation was used through side-
wall vents covered with nets to avoid insect’s pressure.
The temperature range was kept between 25–30°C and
a hand watering-can was used for watering the pots.
After 6 months, different growth parameters such as
number of shoots, number of leaves per shoot, leaf
area, shoot length, plant height and leaf chlorophyll con-
tents of leaves etc. were recorded.
Determination of selected nutrients concentration
in leaves
The nutrients contents of leaves were determined follow-
ing the procedure designated by (Welz and Sperling
1999). Briefly, the mature leaves were collected, ground
and dried by oven for 2 days at 68°C. To determine the
amount of nitrogen, a micro-Kjeldahl was used. For
further analyses H2SO4-Sesalicylic acid mixture was
used to digest the rest of the grinded samples. Indophe-
nol-blue method was used for the quantity analysis of
phosphorus (P) by using spectrophotometry UV-1600
(PC), when it reacts with ascorbic acid. Similarly, the con-
centration of micro-nutrients such as manganese (Mn),
Iron (Fe), zinc (Zn) and copper (Cu) concentrations
were measured by Flame Atomic Absorptions Spec-
trometry method of AOAC (1990). The chlorophyll con-
tents of the leaves were measured by chlorophyll
metre (SPAD-502 konica Minolta, Japan) and 3–4 read-
ings were taken across each leaf. Means of the readings
were determined by internal calculating setup of the
chlorophyll metre (Samsone et al. 2007).
Statistical analysis
For Statistical analysis, the data were subjected to
ANOVA by using software statistics 8.1. and Microsoft
Office Excel 2007. The data presented are from represen-
tative experiments that were repeated at least twice with
similar results. Treatments were compared via ANOVA
using the least significant differences test (LSD) at 5%
(p ≤ 0.05) probability level.
371
Results
Isolation and purification of PGPRs
Total of 95 Plant growth promoting Rhizobacterial
(PGPR) strains were initially isolated from rhizosphere,
rhizoplane and endo-rhizoplane of plum plants from
different localities of district Swat, Pakistan. Among
them only 40 strains were different from each other
while remaining were the replicate of same character-
istics (Supplementary Table 1). These 40 isolates were
re-streaked to purify the colonies for further detail
characterisation.
Morphological characterisation of PGPR
All these 40 selected isolates were screened on the basis
of their colony and cell morphology such as Gram reac-
tion, cell shape, colony colour, colony form, elevation,
margins and colony opacity, as shown in Supplementary
Table 1. Among them, 17 isolates were recorded Gram
negative while remaining 23 isolates were Gram +ve
on the basis of shape, 26 isolates were declared as
cocci and rest of them were observe as rod shaped.
Biochemical characterisation of isolated strains
The ability of all 40 isolates to produce ammonia, sidero-
phore and Hydrogen cyanide (HCN) were tested using
standard procedures as described in material and
method section.
Ammonia
Among all the selected isolates, 17 strains showed strong
positive, seven isolates were observed moderate positive
while twelve strains were noted as weak positive for the
ammonia production. Only four strains (IPL-07, IPL-11,
IPL-20 and IPL-30) were noted negative for ammonia
production (Supplementary Table 2).
Siderophore and HCN
The presence of yellowish zone around the colony was
considered as siderophore positive. Among the 40 bac-
terial isolates, only 3 strains (IPL-07, IPL-09 and IPL-13)
were observed positive for siderophore production
while all other were documented negative. For the pro-
duction of HCN, all isolates were noted negative except
three isolates which responded positive to HCN pro-
duction test. Among these three isolates, IPL-27 was
strongly positive (+++) and IPL-02 was moderately
positive (++) as shown in Table 2 and IPL-05 was
weakly positive (+).
372 I. ALI ET AL.

Table 2. Effect of PGPR on growth of plum plant.

Shoots Number of leaves Per Shoot diameter
Strain’s Code Number shoot Leaf area (cm) (mm) Shoot length (cm) Plant Height (cm)
Control 10.70 A 5.63 A 4.13 A 2.29 A 7.60 A 30.20 A
IPL-02 12.76 B 10.90 BC 5.00 B 3.33 B 12.63 A 36.23 BC
IPL-08 16.80 C 12.53 DE 6.56 CD 3.67 BCDEF 16.63 BC 37.06 BDEF
IPL-09 18.50 DG 10.33 BC 6.68 C 3.91 CD 16.73 BCD 37.70 DE
IPL-10 14.70 E 8.60 F 5.83 EF 3.51 BDEF 14.96 EF 36.23 BG
IPL-13 17.76 D 12.60 DE 6.56 CD 3.91 CD 17.70 BC 37.60 DEF
IPL-17 16.70 C 10.36 BC 5.84 EF 3.77 CDEF 16.13 CDE 36.36 BCF
IPL-18 15.73 F 9.90 CF 6.17 DE 3.52 BDEF 14.70 F 33.73 G
IPL-19 17.80 D 11.60 BE 6.07 DE 3.87 CDE 17.73 BG 36.70 BCEF
IPL-24 16.60 C 8.63 F 5.31 GB 3.47 BEF 15.30 DEF 33.23 G
IPL-27 17.73 D 10.06 C 6.10 E 3.78 CDEF 18.63 GH 37.53 DEF
IPL-29 15.67 F 10.53 BC 6.07 E 3.70 BCDEF 15.96 CDEF 35.43 C
MIX-1 (IPL-9, 13, 17, 19, 27, 18.73 G 12.96 D 6.89 C 4.06 C 19.73 H 38.08 D
29)
MIX-2 (IPL-02, 08, 10, 18, 24) 16.70 C 9.60 CF 5.60 FG 3.40 BF 16.70 BC 35.56 C
LSD (0.05) 0.806 1.325 0.4 0.431 1.328 1.296
Values designated with different alphabets were significantly different. Values with common or same alphabet had no significant difference.
The Effect of selected PGPRs after screening was tested on plum plant growth as shown. The values in same column having different letters are significantly
different at value p < 0.05.

Indole acetic acid (IAA) were found as positive for pectinase production (Sup-
plementary Table 2).
The IAA production ability of all 40 strains were exam-
ined by observing the cherry red ring formation. Out of
these, only three isolates (IPL-17, IPL-19 and IPL-2) were In vitro antagonism of pathogenic fungi by dual
found positive for IAA production, whereas, all others culture assay
were unable to produce IAA (Supplementary Table 2).
All the selected isolates were tested in vitro against the
The concentration of IAA was quantitatively determined,
pathogenic fungal strains. For Fusarium oxysporum, eigh-
the strain IPL-19 produced maximum IAA (13.41 µg/ml)
teen isolates showed antagonistic activity and formed
followed by IPL-17 (11.21 µg/ml) and IPL-2 (6.54 µg/ml).
clear zones with different diameters (Supplementary
Table 2). Among them, only four strains coded IPL-08,
Phosphate solubilisation and motility IPL-10, IPL-13 and IPL-29 showed maximum percent
growth inhibition which ranged from 55 to 78 PGI. Five
For phosphate solubilisation ability, only 28 isolates were
strains IPL-07, IPL-15, IPL-27, IPL-30 and IPL-32 exhibited
capable of solubilising tri calcium phosphate, thus creat-
31–39 PGI while nine other strains coded as IPL-01, IPL-
ing hallow clear zone around the colony. Among 28 posi-
tive, eight strains showed maximum phosphate
solubilisation with solubilisation index between 2 to
2.2.37 (Supplementary Table 2). Remaining 20 strains
had solubilisation index between 1.14–1.88. Similarly,
the most common character of motility for PGPRs iso-
lated strains were also analyzed. Out of 40, only 17
strains were found to harbour the ability of movement
in the media (Supplementary Table 2). The Figure 1
shows the representative of all experimented figures.

Enzyme production capability of selected isolates

PGPRs microbes produce several enzymes such as cata-
lase, oxidase, amylase, protease and pectinase. Our
study revealed that most of the plum roots isolates (n
= 34) were catalase positive while 6 isolates were noted
Figure 1. Phosphate solubilisation on Pikoviskaya agar media by
catalase negative. Similarly, out of 40 strains, 13 strains IPL-18 and IPL-19. The IPL-18 and IPL-19 shows the representa-
were oxidase positive, 27 strains were amylase positive tive figure of all triplicate phosphate solubilisation by selected
30 strains were protease positive, whereas 36 isolates strains of PGPRs.
 ACTA AGRICULTURAE SCANDINAVICA, SECTION B — SOIL & PLANT SCIENCE
02, IPL-09, IPL-14, IPL-17, IPL-21, IPL-24, IPL-25 and IPL-38
showed 12–30 PGI.
Additionally, these 40 isolates were also tested against
the pathogenic Rhizoctonia solani species. Among all, 22
isolates significantly inhibited the growth of Rhizoctonia
solani. Five strains coded IPL-08, IPL-9, IPL-13, IPL-17 and
IPL-29 showed maximum inhibition (ranged between
60–78 PGI), followed by IPL-11 and IPL-27 (50–60 PGI)
while other 15 isolates showed PGI ranging between
10–50 (Supplementary Table 2).
Selection of most efficient strains from forty
selected isolates
Keeping in view the above tested morphological and
biochemical parameters, only 11 most efficient strains
were selected as shown in Table 1. As these strains
were found positive in most of the important test for
PGPR (Supplementary Table 2), hence further carried to
evaluate their impact on growth of one year old young
plum plants under green-house conditions.
Effect of PGPR on growth of inoculated young
plum plants
After keen identification, thirteen treatments of selected
strains (11 isolates + 2 Mix of strains as shown in Table 2)
were applied on roots of plum plants and grown in green
house. After 6 months of inoculation, it was observed
that bacterial inoculation has significantly increased the
growth of plants in terms of the shoot numbers, leaves
number per shoot, shoot diameter, shoot length and
plant height as compared to control (Figure 2 and
Table 2).
Of all 13 treatments, 5 isolates MIX-1, IPL-09, IPL-19,
IPL-13 and IPL-27 showed highest number of shoots
with a range of 17–19 shoots/plant as shown in
Table 2. Six treatments coded IPL-08, IPL-17, MIX-2, IPL-
24, IPL-18 and IPL-29 presented moderate number of
shoots (15–17 shoots/plant) while IPL-2 and IPL-10 dis-
played 12.7 and 14.7 shoots/plant respectively. Similarly,
all other parameter including number of leaves/shoot,
their area and the shoot diameter/length/height were
also increased as compared with control (Table 2). In all
the treatments, Mix-1 showed the highest growth rela-
tive to control as shown in Figure 2, followed by all
others (pictures not shown).
Effects of selected bacteria on leaf nutrients
content
The growth effect of all PGPRs treatments on one year
old young plum plants was also evaluated by
373
Figure 2. Effect of plant growth promoting rhizobacteria (PGPR)
treatments on plum plant growth. The growth characteristics of
plum plant seedlings grown in pots under greenhouse conditions
with selected isolates (Mixed 1 T1) in comparison of control plum
plant (Control T1). The figure shows the representative plum
plant and all the experiments were conducted in triplicates.
determining and comparing the chlorophyll contents
of leaves from inoculated plants and un-inoculated
control plants. Except IPL-29, which slightly decreased
chlorophyll content, the inoculation of plants by all treat-
ments (n = 13) enhanced the concentration of chloro-
phyll (Table 3). The concentrations of N and P were
also significantly increased with respect to control.
Majority of treatments (n = 9) increased Cu content
while 4 of them coded (IPL-02, IPL-10, IPL-24 and IPL-
29) slightly decreased it. Mn was also increased by 11 iso-
lates while only two (IPL-10 and IPL-24) decreased them.
Fe content was increased by all treatments except IPL-10.
Similarly, Zn content was also increased by all the
selected isolates except IPL-17 which slightly decreased
Zn content as compared to control (Table 3). After the
application of these thirteen treatments from 11 selected
strains, 6 strains among them (IPL-08, IPL-09, IPL-13, IPL-
17, IPL-19 and IPL-27) were selected as PGPR for plum
due to their ability of significantly increasing the
growth of plum plants (Tables 2 and 3).
Discussions
Several studies have explored the importance of PGPR
for plant growth and pathological protection. These bac-
teria exist on the surface or inner part of the plant roots
which play important beneficial roles that directly or
indirectly (Lamont and Pérez-Fernández 2016). The
374 I. ALI ET AL.

Table 3. Effects of PGPRs on some nutrients compositions of plum leaves.

Strain’s Code Chlorophyll Contents N (%) p (%) Cu (mg kg1) Mn (mg kg−1) Fe (mg kg−1) Zn (mg kg−1)
Control 35.20 AB 2.58 ABC 0.16 A 0.03 A 0.09 A 1.21 A 0.12 ABC
IPL-02 36.80 C 2.63 ABCD 0.21 A 0.03 A 0.10 A 1.63 BCD 0.14 DEF
IPL-08 41.50 D 2.65 ABCD 0.16 A 0.05 B 0.17 A 1.24 AE 0.12 EF
IPL-09 35.56 A 2.70 AD 0.27 A 0.07 C 0.10 A 1.80 B 0.25 AB
IPL-10 39.60 E 2.65 ABCD 0.17 A 0.03 A 0.09 A 1.17 A 0.15 DEF
IPL-13 40.53 F 2.65 AD 0.17 A 0.05 B 0.10 A 1.66 BC 0.26 AB
IPL-17 37.62 CG 2.74 D 0.18 A 0.05 B 0.13 A 1.65 BC 0.10 F
IPL-18 34.66 B 2.64 ABCD 1.71 B 0.05 B 0.14 A 1.75 BC 0.33 GH
IPL-19 39.40 E 2.66 ABD 0.23 A 0.04 AB 0.10 A 1.22 A 0.25 AB
IPL-24 35.23 AB 2.59 E 0.16 A 0.02 A 0.01 A 1.61 BCD 0.25 AB
IPL-27 41. 76 D 2.63 ABCD 0.20 A 0.03 A 0.19 A 1.43 DE 0.13 DEF
IPL-29 33.26 H 2.59 BCE 0.18 A 0.03 A 0.86 B 1.43 DE 0.17 CDE
MIX-1 38.33 G 2.67 ABCD 0.74 C 0.06 BC 0.76 B 1.56 CD 0.27 AH
MIX-2 35.80 H 2.59 CE 0.22 A 0.05 B 0.18 A 1.70 BC 0.35 G
LSD (0.05) 0.863 2.074 0.564 0.019 4.303 2.447 4.303
Values designated with different alphabets were significantly different. Values with common or same alphabet had no significant difference.
The effect of PGPR on plum plant growth was analyzed on the basis of some nutrient composition measurement as shown in comparison of control plum plant.
The values in same column having different letters are significantly different at value p < 0.05.

effect of PGPR on plant growth is influenced by both
biotic and abiotic factors including bacterial species
and the soil types (Singh and Jha 2016). Therefore, this
study was conducted to explore the isolation and identi-
fication of PGPR from a previously unexplored soil so as
to select the native bacterial strains having a potential to
enhance the growth of plum without the application of
chemical fertilisers and hazardous fungicides.
In this study, 40 positive PGPR were isolated from the
rhizosphere of plum plants from different locations of
Swat region of Northern Pakistan as shown in the Sup-
plementary Table 1. On the basis of Gram staining, 17 iso-
lates were recorded Gram negative while remaining 23
isolates were found Gram +ve. Morphologically, 26 iso-
lates were declared as cocci and rest of them were
observe as rod shaped, which are shown in the Sup-
plementary Table 1. Biochemical screening was per-
formed to assess the ability of PGPRs to produce
ammonia, siderophore and Hydrogen cyanide (HCN) in
accordance with previous studies (Devi et al. 2013). The
efficiency of PGPR for plant growth augmentation is
mainly characterised by ammonia production, which
indirectly influences plants growth (Yadav et al. 2010).
In this study, 17 strains were shown to have the ability
of strong positive ammonia production and the detail
of each strain is shown in Supplementary Table 2. The
second most important characteristic for selection of
most efficient PGPR is the production of siderophore
(Beneduzi et al. 2012). In our screening, three strains
(IPL-07, IPL-09 and IPL-13) were observed positive for
siderophore production while all other were recognised
negative, where the details for each strains are shown
in Supplementary Table 2. It has been reported that
many rhizobacteria produce antifungal metabolites
such as, HCN, phenazines, pyrrolnitrin, 2,4-diacetylphlor-
oglucinol and tensin (Babalola 2010). Considering the
importance of HCN production by rhizobacteria, as
shown in Supplementary Table 2, we also performed
the HCN based selection and only three strains (IPL-27,
IPL-02 and IPL-05) were analyzed as positive.
Another commonly reported PGPR characteristic is
the production of phytohormones such as IAA, which is
considered as a most commonly reported mechanism
for plant growth (Benidire et al. 2016; Liao et al. 2017).
Three isolates showed positive result for the production
of IAA (all the 40 isolates data are shown in Supplemen-
tary Table 2 for detail). Among PGPRs, in a similar passion
the phosphate solubilisation ability of all selected iso-
lates was also determined following the importance of
PGPR for phosphate solubilising capacity (Oteino et al.
2015). As shown in Table 1 and Supplementary Table 2,
eight out of 28 strains showed maximum phosphate
solubilisation with solubilisation index between 2 to
2.2.37. The screening of isolates on the basis of motility
was performed while considering the importance of
motility of efficient PGPR (Vande Broek et al. 1998). The
strains were also tested for their lytic enzyme activity
as shown in Table 1. Our results revealed that most of iso-
lates are positive for catalase, oxidase, amylase, protease
and pectinase activities. Production of lytic enzymes
such as α-amylase, cellulase, pectinase and proteases
are common mechanism used by bacteria to inhibit
growth of the other pathogenic microorganisms
(Dinesh et al. 2015).
In the present study, bio-control activity of selected
isolates was observed against two fungal pathogens
using dual plate culture technique as shown in Table 1
for 11 selected isolates. For pathogenic strains Fusarium
oxysporum and Rhizoctonia solani, 18 isolates showed
antagonistic activity against Fusarium oxysporum while
22 strains showed antagonistic activities against Rhizoc-
tonia solani (Supplementary Table 2). Several previous
 ACTA AGRICULTURAE SCANDINAVICA, SECTION B — SOIL & PLANT SCIENCE
studies have also reported the importance of PGPR’s
antagonistic activities against fungal pathogens (Goes
et al. 2002; Sumesh 2006; Zaki et al. 2006; Du et al. 2016).
Finally, considering all the tested parameters of iso-
lated PGPRs, 11 strains were characterised as most
promising PGPRs and were used to evaluate their
impact on growth of one year old young plum plants
under green-house conditions. Application of two
mixture treatments was also performed due to the
fact that a treatment comprising of more than one
PGPR strain is considered more desirable/effective as
they might have an additive or synergistic effect as
compared to single cultures (Broadway et al. 1998). All
the tested 11 strains along with two random mixed
strains which are represented by mix 1 and mix 2 are
shown in Table 2. The Several previous reports have
indicated that PGPR application enhances the seed ger-
mination and plant growth by increasing the availability
of nutrients, IAA production and or by reducing the inci-
dence of seed mycoflora, which can be detrimental to
plant growth (Da Mota et al. 2008; Salomon et al.
2014; Chowdhury et al. 2015).
The application/inoculation of PGPR strains in our
study significantly enhanced the growth of one-year-
old plum plants through exhibiting various plant
growth promoting traits. Our in vitro experimentation
revealed that selected PGPRs have antagonistic activity
against pathogenic strains Fusarium oxysporum and Rhi-
zoctonia solani, which might be one of the possible
growth induction mechanism of plum plant in vivo. As
shown in Table 2, after six months of inoculation, the
shoot number, leaves number per shoot, shoot diameter,
shoot length and plant height were significantly
increased when compared with control. Our results are
in good agreement to studies conducted in past which
have reported the PGPR’s potency of influencing plant
growth parameters such as plant shoot numbers,
leaves number per shoot, shoot diameter, shoot length
and plant height (Chakraborty et al. 2009; Park et al.
2015; Wang et al. 2015). A similar pattern of result is
also evident in previous studies that reported the
PGPRs effect on the growth, yield and fruit quality of
various fruit plants (Esitken et al. 2003; Akca and Ercisli
2010; Arikan and Pirlak 2016). Similar results for chloro-
phyll contents of leaves as compared to control have
also been obtained as reported in other published
data, which examined the effect of rhizobacterial appli-
cation on plant growth patho-protection and in terms
of influence on chlorophyll contents of leaves (Habib
et al. 2016; Mahmood et al. 2016). As shown in Table 3,
the leaves of the treated plum plant were also analyzed
for selected nutrients compositions in comparison of
control.
375
This study initially screened 11 isolates (among 40
tested) of PGPR from indigenous Pakistani plum plants
root soil system. The results demonstrate that these
strains possess several plant growth promoting traits as
well as in vitro antagonistic activity against pathogenic
strains Fusarium oxysporum and Rhizoctonia solani
which is likely to provide an efficient protection against
pathogenic fungal strains in vivo. A further thorough
screening demonstrates that 6 isolates (IPL-08, IPL-09,
IPL-13, IPL-17, IPL-19 and IPL-27) have shown the best
PGPR characteristics and are most suitable for growth
and yield enhancement of plum. We expect that the iso-
lation and screening of these PGPR would be a step
forward for improved biofertilizer applications and com-
mercial use as biocontrol agents in agriculture sector.
The biofertilizers efficiency of these selected PGPR iso-
lates for plum may be further enhanced with the use
of genetic screening technology and optimisation with
required environmental condition.
Acknowledgements
The authors would like to thank the staff, technical members
and administration of NARC Islamabad, Pakistan for allowing
to conduct this study and providing facilities in Land Resource
Research Institute.
Disclosure statement
No potential conflict of interest was reported by the authors.
Notes on contributors
Izhar Ali is currently enrolled as a PhD student at School of Life
sciences, Lanzhou University, China. He received his M. Phil
degree from the Department of Microbiology, Hazara Univer-
sity Mansehra, Pakistan in 2015. His research interest is to
study the Plant-Microbes and Microbes-Microbes interactions.
Tariq Sultan is serving as a Principal Scientific Officer in Paki-
stan agricultural research council, a prestigious organization
dedicated to provide science-based solutions to agriculture of
Pakistan. He earned his PhD in the field of soil science from
PMAS-Arid Agriculture University, Rawalpindi, Pakistan. He
has completed a number of projects focused on the sustainable
agriculture techniques. He is keen to explore the positive
microbial influence on the growth and health of plants.
Fazli Subhan is an Assistant Professor in the Department of
Microbiology, Hazara University, Mansehra. He has recently
earned his PhD from Pusan National University, South Korea.
He has two international patents in the field of 3D bio model-
ing. His research interests include the utilization of microbes
to produce cost-effective products for various industries.
Kashif Syed Haleem completed his BS(Hons) in Microbiology in
2006 from the Department of Microbiology, Hazara University,
Mansehra and was appointed as Lecturer in the same depart-
ment in 2007. He was awarded with a full-time PhD studentship
376 I. ALI ET AL.
by Higher Education Commission of Pakistan in 2008. After
completion of his PhD from the University of Leicester, UK in
2012, he served as lecturer until January 2016. Currently, he is
serving as Assistant Professor of Microbiology and is actively
engaged in studying the host responses to colonizing
microorganisms.
Nighat Sultana is a full-time Assistant Professor in the Depart-
ment of Biochemistry, Hazara University, Mansehra Pakistan.
She received her M. Phil degree in Biochemistry from Quaid I
Azam University, Islamabad in 2006 and served as Lecturer
prior to leaving for her PhD in 2008 from Exeter University,
UK. After the completion of her PhD in 2012, she has focused
to explore the Biochemical mechanisms of microbes and plants.
Isfahan Tauseef is among the pioneer regular faculty members
of the Department of Microbiology, Hazara University, Man-
sehra. He earned his M. Phil degree from Quaid I Azam Univer-
sity Islamabad in the field of Microbiology in 2006 and soon
after his joining as lecturer Microbiology at Hazara University,
he won a merit-based studentship to pursue his PhD studies
at the University of Leicester, UK. After his return in 2012, he
served as Lecturer until January 2016. He is currently serving
as the Head of Microbiology Department and has interest in
the field of Microbial Molecular Genetics.
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