550
Journal of Basic Microbiology 2011, 51, 550 – 556
Research Paper
Bio-control and plant growth promotion potential
of siderophore producing endophytic Streptomyces
from Azadirachta indica A. Juss.
1
1
2
V. C. Verma , S. K. Singh and Satya Prakash
1
2
Centre of Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University,
Varanasi UP, India
National Facility for Tribal and Herbal Medicine, Institute of Medical Sciences, Banaras Hindu University,
Varanasi UP, India
Three endophytic actinomycetes strains recovered from surface sterilized root tissues of
Azadirachta indica A. Juss. (Meliaceae), plants were selected through tests for their potential as
bio-control and plant growth promoting agents. It was also observed that the seed treated with
the spore suspension of three selected strains of Streptomyces, significantly promoted plant
growth and antagonized the growth of Alternaria alternata, causal agent of early blight disease
in tomato plant. It was observed that the three selected strains prolifically produce IAA and
siderophores that play vital role in promotion of plant growth and in suppression of Alternaria
alternata. Interestingly, Streptomyces strain AzR-051 produced the highest amount of IAA at
13.73 μmol ml–1, compared to strains AzR-049 and AzR-010 9.22 μmol ml–1 and 10.43 μmol ml–1
respectively. It also produces siderophores higher than the other two strains. Thus these
endophytic isolates have the potential as plant growth promoters as well as a bio-control agent,
which is a useful trait for crop production in nutrient deficient soils.
Keywords: Endophytes / Streptomyces / Bio-control / Siderophore / IAA / Azadirachta indica
Received: April 24, 2010; accepted: August 05, 2010
DOI 10.1002/jobm.201000155
Introduction*
Pathogenic micro-organisms are a growing concern
worldwide because of plant health, food production
and ecosystem stability. The growing population puts
immense pressure of raising the agriculture production
to cope with food security around the globe. This ultimately results in intensifying the dependability on
agrochemicals for crop protection from pathogenic
microbes [1], irrespective to their negative consequences i.e. development of resistance towards the
applied agents and their environmental impacts [2].
Furthermore, the growing awareness among consumers
for pesticide free food has led to the search for a substi-
Correspondence: Vijay C. Verma, Centre of Experimental Medicine
and Surgery (CEMS), Institute of Medical Sciences, Banaras Hindu
University, Varanasi 221 005 UP India
E-mail: [email protected]
Phone: 0091 8853953113
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tute of agrochemicals, in controlling the plant pathogenic microbes. Biological control is an alternative for
agrochemicals in not only controlling plant diseases
but also in stabilizing ecosystem. In recent years, several studies on the biological control of plant diseases
have been reported using antibiotic metabolites of microbial origin [3–5]. Many bacterial and fungal strains
such as Pseudomonas, Burkholderia, Trichoderma etc., were
evaluated and reported for having their potential in
promoting plant growth. These microbes colonize the
rhizosphere and plant root as plant growth promoting
rhizobacteria (PGPR) and enhance the plant growth
[6, 7] by increasing availability of nutrients to their
host. In the context of increasing international concern
safe food and environment, the use of PGPR for reducing chemical inputs in agriculture is a potentially important issue, as are microbes that exist in other habitats like endophytes. Endophytes are organisms living
within the tissues of higher plants without causing any
symptoms. These microbes can be isolated from plant
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Journal of Basic Microbiology 2011, 51, 550 – 556
tissues using strict surface-sterilized methods [8]. The
internal tissues of plants provide a relatively uniform
and protected environment when compared with the
rhizosphere and rhizoplane microbes [9]. We are interested in the antifungal activity of endophytic actinomycetes which has been a focus in the exploitation of
these organisms as excellent bio-control as well as plant
growth promoting agents against phytopathogenic
fungi.
Streptomycetes are among the major groups of micro-organism in rhizosphere and soil. They are most
prolific producers of bioactive natural compounds including antibiotics. Besides rhizosphere, endophytic
Streptomyces have been isolated from several plants and
many of them were reported to have intensive in vitro
activity against phytopathogens [10]. Several examples
are reported on the bio-control potential and bioactive
natural products from endophytic Streptomyces. Such as
Streptomyces lydicus WYEC108, which colonizes the nodules of pea plants, increases root nodulation and improves the bacteroids vigour in the nodules. This may
be due to the enhanced nodular assimilation of iron
by the Streptomyces [6]. Endophytic Streptomyces from
Rhododendron produced a novel antifungal antibiotic,
fistupyrone, which protected Chinese cabbage from
infection by Alternaria brassicicola [3]. Some endophytic
Streptomyces synthesize cytokinin-like metabolites that
inhibit rice seed germination and rooting [11]. Thus
more study on these bioactive metabolites will further
our knowledge in exploiting the endophytic Streptomyces as potential bio-control agent, which also resolute our knowledge about endophyte-host relationship.
Early blight is a very common disease of tomato. It
causes leaf spots fruit rot and stem lesions on tomato.
The disease can occur over a wide range of climatic
conditions and can be very destructive if left uncontrolled, often resulting in complete defoliation of
plants. The initial screening of the root tissues along
with the leaf and stems were performed, and a handful
isolates belonging to the genera Streptomyces, Sacchromonospora, Streptosporengium, Microbispora, Nocardia etc.
were recovered [10]. Starting with 62 strains we successfully screen the three potential strains of Streptomyces spp. which shows promising potential in PGP activity. Out of 62 endophytic actinomycetes about 46 were
identified as Streptomyces spp., while other prominent
genera recovered are Sacchromonospora, Streptosporengium, Microbispora, Nocardia etc. from surface sterilization of root tissues of Azadirachta indica plants, sampled
from the local habitat. These strains were chosen to
screen for bio-control activities and three Streptomyces
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Plant growth promotion by endophytes
551
strains showed promising antagonistic activities against
Alternaria alternata the pathogen responsible for early
blight in tomato seeds (var. HS 102). Streptomyces potential to produce a wide array of biologically active compounds including antibiotics and hydrolytic enzymes
additionally they were highly resistant against desiccation and stresses [3, 5], these features make them very
attractive candidate for the bio-control agent [12–14]. In
this study the endophytic Streptomyces spp. from the
‘Neem’ (Azadirachta indica A. Juss.) plants was isolated
and screened for the potential antagonistic activity
against the Alternaria alternata, causal microbe of early
blight disease in tomato plants with their effect on
plant growth promotion.
Materials and methods
Isolation and identification of endophytic
actinomycetes
Endophytic Streptomyces were isolated from Azadirachta
indica A. Juss. The leaf, stem and roots were collected
from the healthy neem plants growing in and around
campus premises of Banaras Hindu University, Varanasi, India. The root and stems were cut in to small
pieces (around 5 cm in length) and rinsed in running
tap water for 5 – 8 min followed by sterile distilled water. After surface sterilization in 75% ethanol (5 min),
the root and stem rods were rinsed three times in
sterilized distilled water, and aseptically cut in to small
pads (0.5 × 0.5 cm2) [4, 15]. The effectiveness of the
surface sterilization procedure was reinforced by the
tissue marker method [16]. The small pads were
carefully placed in to Streptomyces agar (S-agar; containing per litre: 10 g dextrose, 4.0 g casein hydrolysate,
0.5 g K2HPO4, 0.2 g MgSO4 ⋅ 7 H2O, 0.1 g CaCl2 ⋅ 2 H2O,
10 mg ferric citrate, 0.01 mg CoSO4 ⋅ 7 H2O, 0.1 mg
CuSO4 ⋅ 5 H2O, 1.5 mg H3BO3, 0.8 mg MnSO4 ⋅ H2O, 0.2 mg
(NH4)6Mo7O24 ⋅ 4 H2O, 0.6 mg ZnSO4 ⋅ 7 H2O and 6.0 g agar
in 1,000 ml of deionized water.) plates and incubated
at 25 °C for 20 days until the endophytic actinomycetes were discernable. Each isolate was then grown and
examined to ascertain that it originated from a single
spore [5]. The actinomycetes were classified based on
their morphological characteristics on S-agar medium
[17] and the identification of strains were performed
by the methods described previously [10]. Besides morphological characteristics, the utilization of gelatin,
Tween 80, cellulose, gelatin and seven sole carbon
sources (glucose, arabinose, xylose, fructose, sucrose,
rhamnose and manitol) were also used to identify the
strains.
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V. C. Verma et al.
Journal of Basic Microbiology 2011, 51, 550 – 556
In vitro antibiosis assay
In order to determine the potential of isolated strains of
endophytic actinomycetes to produce anti-microbial
compounds against Alternaria alternata, the antibiosis
experiments were carried out. The selected strains of
the actinomycetes were inoculated in the single plates
at their two ends together with the test organism Alternaria alternata and the antagonistic activity was recorded as the inhibition zones (in mm) for each strain
of actinomycetes. Based on the results the most potent
antagonists were selected for further study.
IAA production
Indole acetic acid (IAA) production was assayed calorimetrically using ferric chloride–perchloric acid reagent
(FeCl3–HClO4) as described by Gordon and Weber [21].
The selected strains were cultured in nutrient broth for
24 h on a rotary shaker. After 24 h the culture was
centrifuged at 12000 rpm for 5 min. One ml of the supernatant was added to the 2 ml of the FeCl3–HClO4
reagent, and absorbance was read in UV spectrophotometer at a wavelength of 530 nm. The concentration
of IAA was determined using a standard curve.
Seed treatment and PGP activity
Tomato seeds (var. HS102) were used for the seed germination bioassay. The rolled paper towel assay [18]
was used to evaluate the growth promotion potential of
the selected strains of endophytic Streptomyces. Ten days
old cultures of the selected strains were taken and resuspended in sterile distilled water to get the spore
suspensions. The suspensions were diluted with sterile
distilled water and adjusted to 105 CFU, thereafter the
surface sterilized (sequential dipping in to NaOCl 5% to
3 min followed by ethanol 70%, 1 min) tomato seeds
were soaked in the spore suspension for about 30 min.
For determine the number of spores present on the
seed coat after inoculation seeds was agitated in sterile
distilled water and then an aliquot of resulting suspension was spread to S-agar medium. A negative control
of seeds without any treatment and a positive control
with seeds treated with fungicide Chlorothalonil (1%)
was run parallel to the test experiments. After 30 min
the seeds were filtered from spore suspension, dried
aseptically, and spread on a moistend sterile paper
towel (25 × 30 cm). Twenty seeds from each strain was
rolled in paper towels and replicated three times and
incubated up to 5 days at room temperature. After 5 d
root length, shoot length and seed germination (%)
were observed.
P-solubilization
A qualitative determination of phosphate solubilization
potential of selected strains were performed by inoculating actinomycetes on agar containing precipitated
tricalcium phosphate according to Subba Rao [22]. Selected strains were streaked on the surface of agar
plats. The presence of clear zones around the microbial
colonies was used as indicator for positive phosphate
solubilization after overnight (12 h) incubation.
Siderophore production
Siderophore production was tested qualitatively by
chrome azurol S (CAS) agar as described by Alexander
and Zuberer [19]. The selected strains were sub cultured
onto the King’s B (KB) agar plates in triplicates. The CAS
solution was prepared as described by Schwyn and
Neilands [20] and poured into fresh Petri plates. The
selected strains were then inoculated on the CAS agar
plats and incubated overnight. The change of the mixture color from bluish to orange after overnight incubation indicated the presence of siderophores. This test
was repeated three times.
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Statistical analysis
All bioassays were conducted in triplicate and the mean
± SD was taken for the interpretation of our data to
ensure consistency and reproducibility of the results.
The one way ANOVA and Tukeys multiple range tests
(TMRT) were calculated using SPSS (v.10) to determine
the efficacy of different strains of endophytic Streptomyces, in promotion of root and shoot development.
Results
The endophytic actinomycetes were isolated from stem
and root tissues for Azadirachta indica A. Juss. Strains
belonged to the genera Streptomyces, Sacchromonospora,
Streptosporengium, Microbispora, Nocardia spp. Among
these strains, three Streptomyces strains were found to
have the plant growth promoting activity AzR-010, 049
and 051. The AzR-010 has short spore chain, sometimes
single spores with the smooth ornamentation on the
spores. No diffusible pigments were found and substrate mycelium was almost colorless. Strains AzR-049
and AzR-051 had long chains with at least 10 to 12
spores per chain, substrate mycelium were colorless to
brownish while spores are warty in their ornamentation (Table 1). AzR-010 utilizing the glucose, sucrose,
arabinose and xylose while not utilize mannitol and
fructose, AZR-049 utilizes glucose, fructose and xylose
while not utilize manitol, rhamnose and arabinose.
Strain AzR-051 utilizes mannitol, glucose, arabinose
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Journal of Basic Microbiology 2011, 51, 550 – 556
Plant growth promotion by endophytes
553
Table 1. Comparative study of the three potential strains of Streptomyces spp. for their colony and biochemical characteristics.
Characteristics
Endophytic Streptomyces sp.
Features
AzR-010
AzR-049
AzR-051
Spore chain morphology
No. of spore per chain
Substrate mycelium pigment
Spore surface ornamentation
Diffusible pigments
Sugar utilization (1% w/v)
Mannitol
Sucrose
Glucose
Rhamnose
Fructose
Arabinose
Xylose
Hydrolysis of
Gelatin
Cellulose
Tween 80
IAA production
Siderophore production
P-solubilization
short
single
colorless
smooth
short
∼ 10
brownish
warty
short
10–12
colorless
warty
–
+
+
ND
–
+
+
–
ND
+
–
+
–
+
+
–
+
–
–
+
+
–
+
+
+
–
+
+
+
–
+
+
–
+
+
+
+
+
+
–
–
–
AzR stands for the root samples of Azadirachta indica from where the strain was isolated. ND- Not determined.
and xylose but not rhamnose and fructose (Table 1).
Strain AzR-051 hydrolyse gelatin, cellulose and twin 80,
while strain AzR-010 and AzR-049 doesn’t hydrolyse
gelatin and Tween 80, respectively. All the three strains
had cellulase activity that can be helpful in reducing
fungal infection. Many actinomycetes were reported to
have cellulase activity such as Thermomonospora sp., and
Streptomyces transformant [23, 24]. Strains AzR-049 and
051 were potentially more antagonistic (inhibition
ranges in between 12 to 14 mm) to the Alternaria alternata, than isolate AzR-010 (only 9 mm, p < 0.05), this
suggests that the two strains AzR-049 and 051 have
strong antagonistic activity towards this particular
pathogen (Table 2, Fig. 1). The potential of these strains
was assessed in terms of plant growth promotion (PGP)
activity and it was observed that these strains have
promising potential to be exploited as plant growth
promoting agents since they have PGP activity significantly higher than certain fungicides, for example the
application of fungicide Chlorothalonil 1%, even shows
a reduced growth in root and shoot length as well as in
seed germination (Table 3) as compared to the strain
AzR-051. Among all three strains Streptomyces AzR-051
significantly increased the shoot and root length
and enhanced the seedling germination up to 91%,
which was only 66% in the control seeds. Thus this
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
particular strain has advantage over the rest two in
promoting plant growth. Interestingly, Streptomyces
strain AzR-051 produced highest amount of IAA
production (13.73 μmol ml–1) as compared to AzR-049
(9.22 μmol ml–1) and AzR-010(10.43 μmol ml–1) (Table 4)
that also supports its high PGP activity. Besides IAA
content, this strain (Streptomyces AzR-051) has solubilized phosphates and also produced siderophore significantly higher than other two strains AzR-010 and
AzR-049 (Table 1, Fig. 2).
Table 2. In vitro anti-fungal activity of endophytic Streptomyces
sp. against Alternaria alternata, the causal agent for the early
blight disease in tomato.
Accession no.
Endophytic isolates
of
Zone of inhibition against pathogen
Alternaria alternata (mm)
[mean (n = 3) ±SD]
Streptomyces AzR-010
Streptomyces AzR-049
Streptomyces AzR-051
9.33 ± 1.52a
12.67 ± 2.51b
14.00 ± 2.00b
Streptomyces
The results are mean values of three data sets, (Tukeys test, p < 0.05),
different letters within the same column indicate significant
differences between the treatments.
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554
V. C. Verma et al.
Journal of Basic Microbiology 2011, 51, 550 – 556
Table 3. Effects of the three potential strains of endophytic Streptomyces spp. on the germination and growth of tomato seedlings
(var. HS 102).
Seed treatment
Root length (cm)
Shoot length (cm)
Germination (%)
Streptomyces AzR-010
Streptomyces AzR-049
Streptomyces AzR-051
*Fungicide
Control
6.06 ± 0.41a
7.61 ± 0.43b
7.56 ± 0.33b
6.00 ± 0.14a
5.75 ± 0.39c
7.62 ± 0.20a
8.79 ± 0.17b
8.80 ± 0.21b
7.52 ± 0.34a
6.75 ± 0.37c
84.27 ± 5.01a
88.73 ± 0.38b
92.19 ± 0.73b
81.35 ± 0.35a
66.56 ± 0.56c
* The fungicide used was Chlorothalonil 1%, while control was the untreated seeds. The results are mean values of three data
sets, (Tukeys test, p < 0.05), different letters within the same column indicate significant differences between the treatments.
Table 4. Estimation of IAA production from the three strains of
endophytic Streptomyces sp.
Discussion
Endophytic isolates of
Streptomyces
IAA production
(μmol ml–1)
Streptomyces AzR-010
Streptomyces AzR-049
Streptomyces AzR-051
10.43
9.22
13.73
Endophytic actinomycetes are ubiquitous in natural
environments. It was observed that each plant has its
own endophytic microbial biota that contains not only
actinomycetes but also have fungi and bacteria [10, 25,
26] and these endophytic microbes synthesized products exploitable in the pharmaceutical and agrochemical industries. Although lots of reports are available on
the secondary metabolites from these plant associated
microbes, the biological significance of these metabolites in plant-microbe interaction is always open for
discussion [26–28]. These microbes have also been
found to produce several plant hormones like substances such as toyocamycin, pteridic acid, IAA etc.
[3–7] that have vital roles in the plant growth promotion. With this background we started to work on the
endophytic actinomycetes of Azadirachta indica A. Juss.
in search of novel strains that have plant growth promotion activity. Three endophytic Streptomyces strains
in preliminary screening were found to have very active
in promoting the root and shoot growth as well as seed
germination. In the preliminary characterization studies, the endophytic Streptomyces were cultured on S-agar
medium, and colony characteristics were observed
(Table 1). The strain Streptomyces AzR-051 produces
siderophores more strongly than the two other isolates
and this may possibly be one reason for its PGP activity
[7, 29]. Strains AzR-49 and AzR-51 that produces
siderophores are also having very significant antagonistic activity against the pathogen Alternaria alternata, this
suggests that siderophores of these endophytic Streptomyces species due to having high Fe3+ complexing capacity may be responsible for depriving the pathogen for
iron for its metabolic activity and thus reduce their
growth and survival. While the strain AzR-010 has very
low antagonistic activity and also doesn’t produces
siderophore thus it may be conclude that siderophore is
directly involved into the antagonistic activity (Table 2).
IAA production is another attribute that have very vital
Figure 1. Siderophore productions from two strains of endophytic
Streptomyces sp. AzR-049 and AzR-051, isolated from Azadirachta
indica. The clear zone around the Streptomyces colonies indicates
the secretion of siderophores, as they utilized the iron sources and
clear it. The A and B are the native endophytic strains, while the
C and D are the strains from soil to compare the siderophore
productivity from microbes of different habitat.
Figure 2. Antagonistic activity of endophytic actinomycetes Streptomyces strains AzR-049 and AzR-051 against Alternaria alternata.
Lack of pathogen growth in the area colonized by the actinomycete
is evident in both strains.
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Journal of Basic Microbiology 2011, 51, 550 – 556
role in the PGP potential of these strains. Interestingly
all the three isolates have more or less same potential
in IAA production (Table 4) but the isolate AzR-010 and
AzR-051 has very significant IAA producer and thus
promoting root and shoot growth in plants. It has been
reported that the effect of IAA on plant seedlings are
concentration dependent i.e. low concentration may
stimulate growth while high concentration may be
inhibitory, in addition to this different plant seedlings
shows different responses to the IAA. Thus strain
AzR-051 shows a very good PGP activity because it has
high IAA concentration and the positive seedling response by the plant. These strains have strong phosphate solubilization activity, except isolate AzR-049,
that has limited or no phosphate solubilization activity.
This is interesting because, to reduce the environmental impact on agriculture it is an urgent need to
replace the expensive soluble chemical P-fertilizers [30,
31] by novel, cheaper and more ecological but nevertheless efficient P-fertilizers [32] and in this respect these
phosphate solubilizing strains are of great interest. In
several studies it was reported that Streptomyces spp. has
great P-solubilization potential, such as in case of Streptomyces griseus [33, 34]. Unfortunately the ability of
these selected strains to improve plant growth in
P-deficient soil is not yet well established.
Thus current study come up with strains of endophytic Streptomyces especially Streptomyces AzR-051, are
capable of promoting plant growth and fitness in seed
treatment. This could be related to the ability of these
strains to produce IAA, solubilize P and produce
siderophores that promote root and shoot growth and
seed germination together in addition to developing
resistance against pathogens by antagonize them. These
strains are thus especially suited for amendments in
formulations of bio-fertilizers and bio-control products
for sustainable tomato cultivation. Thus, it is required
that after successful selection of PGP strains, their potential should be evaluated under green house conditions to further study the persistency of the plant
growth promoting potential in the field conditions.
Acknowledgements
VCV extend his thanks to the Council of Scientific and
Industrial Research (CSIR) New Delhi, for financial
assistance to this work (CSIR-09/013(205)/2008/EMR-I,
dt.28-09-2008). Authors also thankful to Dr. Sanjeev
Pandey, Biochemical engineering, IT-BHU, for his help
in biochemical characterization of the endophytic Streptomyces strains for IAA and siderophores production.
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Plant growth promotion by endophytes
555
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