ORIGINAL ARTICLE
Streptomyces rochei SM3 Induces Stress Tolerance in ChickpeaAgainst Sclerotinia sclerotiorum and NaClSmita Srivastava1, Jai Singh Patel2, Harikesh Bahadur Singh1, Asha Sinha1 and Birinchi Kumar Sarma1
1 Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India
2 Department of Botany, Banaras Hindu University, Varanasi 221005, India
Keywords
ERF transcription factor, induced systemic
resistance, lignifications, salinity, Sclerotinia
sclerotiorum, Streptomyces rochei
Correspondence
B. K. Sarma, Department of Mycology and
Plant Pathology, Institute of Agricultural
Sciences, Banaras Hindu University, Varanasi,
India.
E-mail: [email protected]
Received: June 16, 2014; accepted: November
5, 2014.
doi: 10.1111/jph.12358
Abstract
Understanding on actinomycetes-mediated stress tolerance in plants is
very limited. This study demonstrated for the first time some stress toler-
ance mechanisms in chickpea via mediation of an actinomycetes strain
Streptomyces rochei SM3. Here, we used the strain SM3 for treating chickpea
seeds and plants raised from such seeds were challenged with Sclerotinia
sclerotiorum and NaCl. Chickpea mortality due to Sc. sclerotiorum infection
was suppressed by nearly 48%, and biomass accumulation was increased
by nearly 20% in the salt-stressed condition in SM3-treated plants com-
pared to non-treated plants. Physiological responses in chickpea under the
challenging conditions showed that phenylalanine ammonia lyase activi-
ties increased in SM3-treated plants. This is followed by accumulation of
higher concentrations of phenolics that led to enhanced lignifications in
SM3-treated plants compared to non-SM3-treated plants challenged with
the same stresses. Antioxidant activities, as assessed through catalase
activities and proline accumulation, also increased in SM3-treated plants
challenged with both the stresses compared to non-SM3-treated plants.
Investigation at genetic level further showed that the strain SM3 triggered
the ethylene (ET) responsive ERF transcription factor (CaTF2) under the
challenged conditions. Thus, from this study, we conclude that actinomy-
cetes St. rochei SM3 trigger the ET-mediated defence pathway in chickpea
and activates the phenylpropanoid pathway for alleviating the stresses
caused by Sc. sclerotiorum and salt in chickpea.
Introduction
Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan
necrotrophic fungal pathogen with a broad range of
hosts worldwide. The pathogen and its related species
cause numerous stem rots, soft rots and wilts of horti-
cultural and agricultural crops including chickpea
(Cicer arietinum L.) (Fuhlbohm et al. 2003). Severe
outbreaks of soft rot can cause yield loss as high as
100%. The pathogen survives in soil as hard sclerotia
for several years and the sclerotia germinate carpo-
genically to produce apothecia. On maturity, apothe-
cia releases large amount of ascospores that spread
over a long distance and serve as primary source
of inoculum for infection. Hence, managing such
pathogens sustainably is a challenge. With the alarm-
ing harmful effect of chemical pesticides, alternatives
are being exploited to manage Sc. sclerotiorum (Sarma
et al. 2007). Biological control, a component of
sustainable crop disease management strategies, is
currently seen as the most potential management
strategy for crop pathogens through reducing patho-
gen inoculum in soil (Lopez-Lima et al. 2013). Antag-
onistic activities of the biocontrol agents (BCAs) once
seen to be the most effective strategy for biological
management of crop pathogens. However, recently, it
is realized that BCAs with antagonistic properties
alone are not sufficient to manage pathogens such as
Sc. sclerotiorum. One of the reasons is explained to be
the improbability of the BCAs to come in direct
J Phytopathol 163 (2015) 583–592 � 2014 Blackwell Verlag GmbH 583
J Phytopathol
contact with the infection causing ascospores of
Sc. sclerotiorum. Therefore, BCAs capable of stimulat-
ing induced systemic resistance (ISR) responses in
hosts have a greater chance to lower the infection
level of Sc. sclerotiorum incited through airborne as-
cospores. Similarly, abiotic stresses are also increasing
globally and causing grave concerns to future agricul-
ture. It is reported that a large area of cultivable soil is
becoming saline every year globally and estimated
that approximately 50% of the arable land may be
affected by salinity stress by the year 2050 (Munns
and Tester 2008). With lack of suitable cultivars to
grow currently under saline conditions in many agri-
cultural crops, alternatives are being sought to address
this issue (Porcel et al. 2012). Microbe-mediated
salinity tolerance can be an effective way to tackle the
situation as some microbes are able to induce systemic
tolerance (IST) in plants against a variety of abiotic
stresses (Yang et al. 2009).
Microbe-induced systemic tolerance in plants has
gained considerable attention recently. Systemic tol-
erance occurs when plants develop enhanced defen-
sive capacity in response to an appropriate signal
perception from pathogens or abiotically challenging
environments. Such perceptions lead to development
of resistance in spatially distant plant parts to specific
pathogens (ISR) and abiotic stresses (IST). These per-
ceptions by plants give rise to exaggerated immune
responses. Therefore, identification, characterization
and utilization of such microbes for possible allevia-
tion of stresses in plants have always considered
important. Previous reports demonstrated that plant
growth-promoting rhizobacteria (PGPR) can increase
plant’s tolerance to soilborne pathogens (Singh et al.
2003, 2013a) and salt stress (Mayak et al. 2004; Yang
et al. 2009). The PGPRs can also promote plant
growth directly or indirectly by mechanisms such
as secretion of 1-aminocyclopropane-1-carboxylate
(ACC) deaminase (Glick et al. 2007), phytohormones
like indole-3-acetic acid (IAA) and production of anti-
fungal enzymes like chitinases (Shoresh et al. 2010),
stimulation of the host phenylpropanoid pathway
(Singh et al. 2013a), stimulation of antioxidants
(Singh et al. 2013b), etc.
Ethylene (ET) is a very important phytohormone,
which participates in major developmental pro-
cesses, including seed germination, cell elongation,
flowering, fruit ripening, organ senescence, abscis-
sion and responses to stresses. Physiologically, ET is
captured by its receptors resulting in the expression
of secondary transcription factors such as ethylene
responsive factors (ERF) (Zhang et al. 2009). ERF
proteins are a subfamily of APETALA2 (AP2)/ET
responsive-element-binding protein (EREBP) tran-
scription factor family and shown to mediate a
variety of stress responses in plants. ERF proteins
share a conserved 58–59 amino-acid domain (ERF
domain) that can bind to two similar cis-elements:
the GCC box, which is found in several pathogene-
sis-related (PR) gene promoters where it confers ET
responsiveness and the C-repeat (CRT)/dehydration-
responsive element (DRE) motif, involved in both
dehydration- and low-temperature responsive gene
expression (Singh et al. 2002).
Several beneficial microorganisms are reported to
enhance defence mechanisms in plants against a
number of pathogens and abiotic factors by activating
defence genes via jasmonic acid (JA)- and ET-medi-
ated signalling pathways (Van der Ent et al. 2009a).
Among the microbial species, the members of Strepto-
mycetes are shown to possess multiple characteristics
which had recently been revealed in several studies
(Hamdali et al. 2008; Franco-Correa et al. 2010).
Among them, Streptomyces rochei is shown to produce
antimicrobial compounds for biological control of soil-
borne pathogens (Anukool et al. 2004), Streptomyces
lydicus to suppress fungal root and seed rots (Yuan and
Crawford 1995), Streptomyces sp. to alleviate salt stress
in tomato (Palaniyandi et al. 2014), Streptomyces viola-
ceusniger to suppress wood-rotting fungi of trees (She-
khar et al. 2006), etc. However, there is no evidence
of host mediation of stress tolerance either at bio-
chemical or genetic levels triggered by actinomycetes
in the earlier reports. Keeping the growing impor-
tance of actinomycetes in agricultural context, the
current investigation was performed with the objec-
tive to assess the mechanisms of host-mediated resis-
tance by an actinomycetes strain St. rochei SM3
against the stresses caused by the pathogen Sc. sclero-
tiorum and salinity caused by NaCl.
Materials and Methods
Bioagent
Soils and decomposed cow dung from different agri-
cultural fields were collected from Varanasi, Uttar Pra-
desh, India. White and grey colonies of Streptomyces
spp. were isolated from actinomycetes isolation agar
(AIA) supplemented with cycloheximide and purified
on International Streptomyces Project medium 2
(ISP-2) slants. Morphological and biochemical charac-
terization was performed and results were compared
with the manual of International Streptomyces Project
(Shirling and Gottlieb 1966). Biochemical tests such
as catalase, H2S production, indole and methyl red
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Actinomycete induce tolerance in chickpea S. Srivastava et al.
test along with gelatin hydrolysis, urea hydrolysis and
chitinase production were also performed following
standard protocols. Melanin production was tested on
ISP-7 (Tyrosine Agar) medium, and colour change
was observed after 1 week of incubation at 30°C(Shirling and Gottlieb 1966). Utilization of different
carbon sources like D-glucose, D-fructose, sucrose,
I-inositol and D-mannitol by the actinobacterial strain
was evaluated on ISP-9 medium (Pridham and Gott-
lieb 1948). The results of morphological and cultural
characteristics were described in Srivastava (2012).
Salt tolerance was tested at different concentrations of
sodium chloride (NaCl: 2–12%) in ISP-2 medium, and
IAA production was determined according to Bano
and Musarrat (Bano and Musarrat 2003), and chitin-
ase activity was assessed according to Mane and Desh-
mukh (2009). Analytical grade reagents were used
from HiMedia (Mumbai, India) throughout the exper-
iments.
Antimicrobial activity of 15 selected strains was
determined on ISP-2 medium by dual-culture
method. The experiment was repeated twice with five
replications for each strain and per cent growth inhi-
bition was calculated. Based on antifungal activities
(growth inhibition and chitinase production) and IAA
production, the actinobacterial strain SM3 was
selected for further studies. Streptomyces rochei strain
SM3 was originally isolated from decomposed cow
dung from an agricultural farm of Varanasi, India.
Morphological and cultural characteristics along with
16S rDNA sequence revealed the identity of the Strep-
tomyces strain and the sequence was deposited in the
GenBank with the accession no. JN128892, and the
pure culture of the strain SM3 was deposited in
National Bureau of Agriculturally Important
Microbes (NBAIM), Mau Nath Bhanjan, India with
the accession no. NAIMCC- B-01002. 1-Aminocyclo-
propane-1-carboxylic acid (ACC) deaminase activity
of the actinobacterial strain SM3 was determined
according to Dworkin and Foster (1958), and the ACC
deaminase gene was amplified using the forward and
reverse primers 50-CACCCTCGTCAGCATCGGAG-30
and 50-AGCTGTCCTTCACGCCGATG-30, respectively,
through PCR (Techne, Stone, Staffordshire, UK).
Experimental set-up
Chickpea cv. Avrodhi seeds were surface sterilized
(1% NaOCl) and divided in two parts – one part was
soaked in 0.1% carboxymethyl cellulose (CMC) and
other part in 0.1% CMC containing cell suspension of
St. rochei SM3 (6.8 9 107 CFU/ml) and kept over-
night. Both treated and untreated seeds were sown in
pots (5 seeds/pot) in a glasshouse. After 20 days of
sowing, each set of pots were subdivided in two sets –Sc. sclerotiorum was applied by placing a 5 mm myce-
lial block of actively growing culture at the collar
region of the stem in one set and NaCl (50, 100, 150
and 200 mM) in the other by pouring 100 ml/pot. Ten
replications were maintained for each treatment and
each pot comprised of five plants. Disease severity
index (DSI) was calculated (Sherwood and Hagedorn
1958) after 4 weeks of pathogen inoculation with
slight modifications. Disease classes: 0 = no symp-
toms; 1 = only lateral branches showing lesions;
2 = lesions on the main stem but without plant death
at the sampling time; 3 = lesions on the main stem
resulting in plant death at the sampling time. Dry
weight was calculated after 4 weeks of NaCl applica-
tion.
Biochemical assays
The defence-response indicators viz., phenylalanine
ammonia lyase (PAL) activity was assessed according
to Havir (1987), total phenolics concentration (TPC)
according to Sarma et al. (2002) and proline accumu-
lation according to Bates et al. (1973). Similarly,
activity of the antioxidant enzyme catalase was
assayed according to Teranishi et al. (1974). For ligni-
fication assay, the stem samples were collected at
96 h after inoculation with the pathogen Sc. sclerotio-
rum and NaCl (200 mM). Fine transverse sections
were observed under light microscope (Nikon DS-fi1,
Tokyo, Japan) after placing a drop of a solution of
0.1 g of phloroglucinol prepared in 10 ml of 95% eth-
anol. Subsequently, when the solution dried, a little
amount of 25% HCl was diffused from the edge of the
cover slip. The appearance of red–violet colour indi-
cated deposition of lignin.
The whole experiment was repeated once and all
assays were carried out in triplicate. The data from
each replication were pooled for calculation of means.
Analytical grade reagents were used from HiMedia
throughout the experiments.
Expression study of a biotic and abiotic stress
responsive ERF transcription factor
Fresh chickpea leaves were collected at 6 h after
stress (Sc. sclerotiorum and NaCl) applications, total
RNA was extracted (Qiagen-Rneasy Plant Mini Kit,
Maryland, USA), and cDNA was synthesized by using
reverse transcriptase (MP Biomedicals, Mumbai,
India). Finally, sterile distilled water was added to
make up the volume to 20 ll. For RT-PCR assay, we
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S. Srivastava et al. Actinomycete induce tolerance in chickpea
used annotated sequences corresponding to gi num-
bers 169746437, 169743966 and 88193282 (available
at NCBI). The expression of the ERF transcript in
chickpea leaves was analysed by a semi-quantitative
PCR (Techne) technique using gene-specific primers,
and the technique was verified with the expression of
ubiquitin using specific primers in a separate PCR. A
forward (50-ACGGTGTTCAGCACTCACAG-30) and reverse (50-ATCTCCACCGAGGAAAGATG-30) gene (CaTF2)-specific primer pair was
used to amplify the ERF transcript in different treat-
ments. A second set of primers (forward 50-GCTACTCCCAATCCCACTC-30 and reverse 50-AATACTT-CATTTCCATCCTGTCC-30) was designed to amplify a
fragment of 168 bp of the C. arietinum ubiquitin gene
(GenBank accession No. CAC12987). The end point
semi-quantitative RT-PCR assays were carried out fol-
lowing the conditions: initial activation at 95°C for
15 min, followed by 35 cycles at 94°C for 1 min, 50°Cfor 1 min and 72°C for 1 min. Final extension was
done at 72°C for 5 min.
Statistical analysis
Statistical analyses were performed with SPSS 16.0
(SPSS Inc., Chicago, IL, USA). Data for chitinase and
IAA were analysed by one-way ANOVA. Mean separa-
tions were performed by Duncan’s multiple range
tests. Differences at P ≤ 0.01 were considered to be
significant. Similarly, data for dry weight, catalase,
PAL, TPC and proline were analysed by two-way ANO-
VA. A multivariate statistical analysis was performed at
P ≤ 0.01 probability level to detect significance
between the interacting factors in both pathogen (bio-
tic) and NaCl (abiotic) applied conditions between
SM3-treated and untreated groups.
Results
SM3 suppresses Sclerotinia soft rot and promotes
biomass under salinity
All actinobacterial strains suppressed mycelial growth
of Sc. sclerotiorum, but highest suppression was
observed in St. rochei SM3 (approximately 74%) (Fig-
ure S1a). Higher growth inhibition by the actinobac-
terial strains may be correlated with high chitinase
production (0.18 U/ml) by the strains (Figure S1b).
Similarly, all actinomycetes strains produced IAA pro-
duction with varied degree and among the 15 selected
strains four viz., SM3, SM11, SM12 and SM13, were
high IAA producers (>40 lg/ml fresh wt.) (Figure
S1c). SM3-treated plants also reduced plant mortality
by 48% compared to non-SM3-treated plants (Figure
S1d). Chickpea biomass varied in different NaCl-trea-
ted plants and biomass decreased (<600 mg/plant)
with increase in NaCl concentration with significant
reduction (<500 mg/plant) at 200 mM NaCl. Interest-
ingly, biomass of chickpea under NaCl stress increased
(>600 mg/plant) in the SM3-treated plants and most
significantly (>550 mg/plant) at the highest NaCl con-
centration (200 mM) (Figure S1e,f). Streptomyces rochei
SM3 was able to tolerate NaCl up to 6% and its
growth inhibited at 8% NaCl (data not shown). Good
growth of SM3 in DF plates indicated positive ACC
deaminase activity. The same was further confirmed
by the amplified PCR product targeted to amplify the
ACC deaminase gene from SM3 (Figure S1g).
SM3 stimulates phenylpropanoid and antioxidant
activities and proline accumulation
Catalase activity in the pathogen-treated plants
without SM3 application showed a declining trend
over the sampling period (Fig. 1A-a). However, in
SM3-treated plants challenged with the pathogen,
the catalase activity increased and reached highest
at 48 h after pathogen challenge. Similarly, catalase
activity was also increased initially (24 h) in all
NaCl-applied plants and then declined rapidly
in the SM3 non-treated plants, whereas in the
SM3-treated plants, its activity increased consis-
tently over the sampling period (Fig. 1A-b). Simi-
larly, in the Sc. sclerotiorum challenged experiment,
PAL activity increased in the SM3-treated plants
following challenge with the pathogen compared to
either only pathogen-challenged plants without
SM3 treatment or only SM3-treated plants without
pathogen challenge (Fig. 1B-a). The activity was
highest at 48 h which declined thereafter at 72 h.
The pathogen-challenged plants without SM3
treatment also showed increased PAL activity over
the control; however, the activities were lower
compared to only SM3-treated plants. In the NaCl-
challenged experiment also, PAL activities were
increased compared to control (Fig. 1B-b). PAL
activity increased with NaCl concentrations and was
highest at 200 mM. However, PAL activities were
relatively high throughout in SM3-treated plants
compared to SM3 non-treated plants in the same
concentrations of NaCl. Total phenolics accumulation
also followed a similar trend with PAL activity
(Fig. 1C-a). However, phenolics accumulation in
pathogen-challenged plants with SM3 treatment and
only SM3-treated plants without pathogen challenge
were almost same. Phenolics accumulation was also
J Phytopathol 163 (2015) 583–592 � 2014 Blackwell Verlag GmbH586
Actinomycete induce tolerance in chickpea S. Srivastava et al.
increased in NaCl treatments. Phenolics content
was more in NaCl-treated plants treated with SM3
compared to NaCl-treated plants without SM3
(Fig. 1C-b). Phenolics accumulation increased with
increase in NaCl concentrations irrespective of SM3
treatment. Thus, activation of the phenylpropanoid
(A) a25
20
Cat
alas
e (µ
M H
2O2
oxid
ised
m
in/g
/FW
) 15
10
5
024 h 48 h 72 h
Ss
SM3
Ss + SM3
Control
8
PA
L (µ
M T
CA
g–1
FW
)
0
1
2
3
4
5
6
7
PA
L (µ
M T
CA
g–1
FW
)
0
1
2
3
4
5
6
7
1
TP
C (
mM
GA
E g
–1 F
W)
Pro
line
(µM
g–1
FW
)
Pro
line
(µM
g–1
FW
)
0.2
0.1
0
0.3
0.25
0.15
0.05
0
0.1
0.2
0.25
0.15
0.05
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
24 h 48 h 72 h
24 h
1st week 2nd week 3rd week 1st week 2nd week 3rd week
48 h 72 h
Ss
SM3
Ss + SM3
Control
Ss
SM3
Ss + SM3
Control
Ss
SM3
Ss + SM3
Control
25
30
20
Cat
alas
e (µ
M H
2O2
oxid
ised
m
in/g
/FW
)
15
10
5
0
24 h 48 h 72 h
SM3+ SM3– SM3+ SM3– SM3+ SM3–
Control
Nacl 50
Nacl 100
Nacl 150
Nacl 200
24 h 48 h 72 h
SM3+ SM3– SM3+ SM3– SM3+ SM3–
Control
Nacl 50
Nacl 100
Nacl 150Nacl 200
TP
C (
mM
GA
E g
–1 F
W)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
24 h 48 h 72 h
SM3+ SM3– SM3+ SM3– SM3+ SM3–
SM3+ SM3– SM3+ SM3– SM3+ SM3–
Control
Nacl 50
Nacl 100
Nacl 150Nacl 200
Control
Nacl 50
Nacl 100
Nacl 150Nacl 200
b
a b
ab
a b
(B)
(C)
(D)
Fig. 1 (A) Catalase activity in chickpea under (a) Sclerotinia sclerotiorum and (b) NaCl stress alone or in combination with Streptomyces rochei SM3.
Results are expressed as means of three replicates and vertical bars indicate standard deviations of the means. (B) Phenylalanine ammonia lyase
(PAL) activity in chickpea under (a) Sc. sclerotiorum and (b) NaCl stress alone or in combination with St. rochei SM3. Results are expressed as means
of three replicates and vertical bars indicate standard deviations of the means. (C) Total phenolic accumulation in chickpea under (a) Sc. sclerotiorum
and (b) NaCl stress alone or in combination with St. rochei SM3. Results are expressed as means of three replicates and vertical bars indicate standard
deviations of the means. (D) Proline accumulation in chickpea under (a) Sc. sclerotiorum and (b) NaCl stress alone or in combination with St. rochei
SM3. Results are expressed as means of three replicates and vertical bars indicate standard deviations of the means.
J Phytopathol 163 (2015) 583–592 � 2014 Blackwell Verlag GmbH 587
S. Srivastava et al. Actinomycete induce tolerance in chickpea
pathway was evident through higher PAL activities
and accumulation of phenolics in SM3-treated
plants compared to the SM3 non-treated plants.
Proline accumulation also varied in pathogen- and
NaCl-challenged plants either treated with SM3 or
not (Fig. 1D-a). Although proline accumulation was
slightly high in SM3-treated plants challenged with
the pathogen compared to the non-SM3-treated path-
ogen-challenged plants, but the differences were not
significant. Proline content in only SM3-treated plants
without pathogen challenge showed only a basal level
increase. However, NaCl-applied plants grown from
SM3-treated seeds showed high proline accumulation
compared to non-SM3-treated plants (Fig. 1D-b). The
increase in proline content was highest at 100 mM
NaCl treatment compared to the two higher concen-
trations of NaCl (150 and 200 mM). The same trend
was also evident in plants treated with NaCl but
without SM3 treatment. Proline accumulation in
NaCl-challenged plants pretreated with SM3 was
significantly high compared to the plants without
SM3 treatment. This is a distinction from the patho-
gen-challenged plants where the differences were not
significant.
Statistical significance
Multivariate analysis of the SM3 treated and
untreated groups of both biotically and abiotically
challenged chickpea plants for the parameters dry
weight, catalase, PAL, TPC and proline are presented
in Table 1. In the Table, bold data represent signifi-
cance of SM3 over treatments without SM3. Only in
few treatments from both groups (SM3 and without
SM3) have no significant differences, but the numeri-
cal values in such treatments are slightly higher than
the control values. Across treatments, 24 and 72 h of
PAL and first week of Proline are not significant in
SM3 group with pathogen stress. In NaCl stress exper-
iment, catalase at 24 h and PAL at 48 h showed insig-
nificant results. From the analysis, we can conclude
that across the treatment groups, SM3-treated group
is showing significant effect in both biotic and abiotic
stress conditions.
SM3 induces lignification
Histochemical staining of stem sections from different
treatments showed significant variations in lignin
deposition (Fig. 2). Highest and uniform lignin depo-
sition in vascular bundles was found in treatments
with SM3 either challenged with the pathogen Sc. scle-
rotiorum or NaCl (200 mM). Phloem and interfascicu- Table
1Multivariate
analysisofboth
Sclerotinia
sclerotiorum-andNaCl-appliedchickp
eaplants
treatedwithorwithoutStreptomycesrocheiSM3
Groups
Dry
weight
Catalase
PAL
TPC
Proline
24h
48h
72h
24h
48h
72h
24h
48h
72h
1st
week
2ndweek
3rd
week
Sc.
sclerotiorum
SM3treated
F1,1=6.3,c
F1,1=731,c
F 1,1=190,c
F 1,1=75,b
F 1,1=00,a
F1,1=111,c
F1,1=13,a
F1,1=105,c
F1,1=34,b
F1,1=25,b
F 1,1=11,a
F 1,1=193,c
F 1,1=198,c
SM3
non-treated
F 1,1=26,b
F 1,1=5,a
F 1,1=1,c
F 1,1=6,a
F 1,1=39,b
F1,1=20,a
F1,1=20,a
F1,1=4,a
F1,1=126,c
F1,1=271,c
F 1,1=273,c
F 1,1=202,c
F 1,1=3,a
NaCl
SM3treated
F 1,4=233,c
F 1,4=4,a
F 1,4=31,c
F 1,4=31,c
F 1,4=41,c
F1,4=4,a
F1,4=30,c
F1,4=184,c
F1,4=48,c
F1,4=37,c
F 1,4=113,c
F 1,4=232,c
F 1,4=146,c
SM3
non-treated
F 1,4=4,c
F 1,4=3,a
F 1,4=38,c
F 1,4=1,a
F 1,4=6,a
F1,4=13,c
F1,4=39,c
F1,4=597,c
F1,4=115,c
F1,4=252,c
F 1,4=34,c
F 1,4=22,c
F 1,4=27,c
PAL,phenylalanineammonialyase;TPC,totalp
henolicsco
ncentration.
Where
a=notsignificant(P
>0.01),b=P<0.01,c=P<0.001,subscript1and4are
interceptanddegreeoffreedom,respectively.
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Actinomycete induce tolerance in chickpea S. Srivastava et al.
lar cambial cells were also found to be highly lignified
in the same treatments comprising SM3. Lignification
in only pathogen-challenged plants without SM3;
NaCl-applied plants without SM3, and only SM3-trea-
ted plants were not high and comparable with the
control.
SM3 triggers ERF transcript CaTF2
The expression of the ERF transcript CaTF2 under
both biotic (pathogen) and abiotic (NaCl) stressed
condition was observed at 6 h after stress applica-
tions. Its expression profile revealed that during
pathogen stress when the plants were treated with
SM3, the expression of the ERF transcript was more
compared to non-SM3-treated plants (Fig. 3). Single
application of SM3 without any stress showed very
low expression of the transcription factor. Similarly,
in NaCl-applied plants, expression of the transcript
was uniform in all NaCl concentrations where the
plants were treated with SM3 compared to the plants
without SM3 treatment. In the SM3 non-treated
plants, the expression of CaTF2 was very poor partic-
ularly at high NaCl concentrations (150 and
200 mM).
Discussion
In the present study, we used an actinomycete repre-
sentative strain St. rochei SM3 to study the potentiality
of actinomycetes-mediated biological control of
Sc. sclerotiorum and NaCl stress tolerance in chickpea.
We also investigated the host-mediated induction of
defence responses in chickpea against the biotic and
abiotic stresses following treatment with the actino-
mycete strain SM3. The strain SM3 suppressed disease
development by Sc. sclerotiorum and significantly com-
pensated for the loss in biomass in chickpea due to
NaCl stress. Biomass compensation by SM3 in chick-
pea plants under the challenge of NaCl may be attrib-
uted to ACC deaminase activities of SM3 through
lowering down the ACC levels leading to lowering
(a) (b) (c)
(d) (e) (f)
Fig. 2 Lignin deposition in chickpea under Sclerotinia sclerotiorum and NaCl stress alone or in combination with Streptomyces rochei SM3. (a) Con-
trol; (b) Sc. sclerotiorum; (c) Sc. sclerotiorum + SM3; (d) SM3; (e) NaCl 200 mM; (f) NaCl 200 mM + SM3.
300 bp
Ladd
er
T1 T2 T3 T4 T5 (50
)
T5 (10
0)
T5 (15
0)
T5 (20
0)
T6 (10
0)
T6 (15
0)
T6 (20
0)
T6 (50
)
100 bp
CaTF2
Ubiquitin
Fig. 3 Expression analysis of an ERF transcription factor CaTF2 through semi-quantitative RT-PCR with chickpea cDNA of Sclerotinia sclerotiorum and
NaCl-applied seedlings either or not treated with Streptomyces rochei SM3. Treatments include: T1 – Control; T2 – Sc. sclerotiorum; T3 – Sc. sclerotio-
rum + SM3; T4 – SM3; T5 – SM3-treated plants with different concentrations of NaCl (50, 100, 150 and 200 mM); T6 – SM3 non-treated plants with dif-
ferent concentrations of NaCl (50, 100, 150 and 200 mM). ERF, ethylene responsive factors.
J Phytopathol 163 (2015) 583–592 � 2014 Blackwell Verlag GmbH 589
S. Srivastava et al. Actinomycete induce tolerance in chickpea
down of ET concentration. Growth compensation in
plants by ACC deaminase producing plant-associated
bacteria is elaborated by Glick (2014). It is a well
known fact that reactive oxygen species (ROS) is gen-
erated during infection by necrotizing pathogens and
abiotic necrosis-inducing agents. However, develop-
ment of necrosis under these situations can be
delayed through induction of antioxidant activities
(Barna et al. 2003). As catalase helps in maintaining
ROS homoeostasis during biotic and abiotic stress,
higher catalase activities in the SM3-treated plants is
an indication of the stimulus generated by SM3 for
high antioxidant activities in the SM3-treated plants.
Biochemical assays further revealed that enhanced
stimulation of the phenylpropanoid pathway took
place in SM3-treated plants under the stressful condi-
tions. Plant phenolics are natural products formed by
activation of the phenylpropanoid pathway and play
a major role in inducing microbe-mediated ISR
responses (Sarma et al. 2002; Lavania et al. 2006).
Separate studies have also demonstrated that benefi-
cial soil inhabiting microbes can stimulate the phenyl-
propanoid pathway in host plants either challenged
with a pathogen or an abiotically stressful condition
(Singh et al. 2003). The present study clearly demon-
strated capability of SM3 to induce PAL activity and
subsequent accumulation of phenolics in chickpea
under both biotic and abiotically stressful conditions.
Similarly, it is well described that under stress condi-
tions, many plant species accumulate proline as an
adaptive response to adverse conditions and is gener-
ally believed that the increase in proline content fol-
lowing stress injury is beneficial for the plant cell.
Proline accumulation during different stressed condi-
tions such as high salinity, drought and biotic factors
was reported earlier (Mattioli et al. 2009). Apart from
the osmoprotectant role, proline also acts as a hydro-
xyl radical scavenger (Schobert and Tschesche 1978).
Chen and Dickman (2005) demonstrated that proline
can function as a potent antioxidant to scavenge ROS
generated intracellularly and thereby inhibit ROS-
mediated adverse cell functions apart from its well-
established role as an osmolyte. Similarly, in many
plant species, proline accumulates in response to envi-
ronmental stresses and acts as a signal molecule to
modulate mitochondrial function, influence cell pro-
liferation or cell death and trigger specific gene
expression, which can be essential for plant recovery
from stresses (Szabados and Savoure 2010). Increase
in proline content in the present investigation during
the sampling period in the SM3-treated plants under
NaCl stress thus appear to be a positive correlation
with the activities of other antioxidants such as
catalase. It can be presumed that proline also behaved
as antioxidant under the influence of the actinomyce-
tes strain SM3 in chickpea and contributed to lower-
ing down the oxidative damage due to NaCl.
Lignin, a polymer of phenylpropanoid compound,
is the last product of the pathway and a strong physi-
cal defence structure that act as a barrier during path-
ogen attack. It contributes to the structural integrity
of xylem vessels as an adaptation mechanism in resist-
ing the stress imposed by salinity (Cachorro et al.
1993). However, its content and composition is
known to change when plants are exposed to various
stresses. An increase in lignification is often observed
in response to attack by pathogen and is believed to
represent one of a plethora of mechanisms adopted to
block pathogen invasion due to its highly non-degrad-
able and antimicrobial nature (Singh et al. 2003,
2013b; Rogers and Campbel 2004). In the present
investigation, it was observed that lignifications
increased in plant tissues under both biotic and abiotic
stress conditions, but lignifications was higher in the
presence of the actinobacterium strain SM3 compared
to the SM3 non-treated plants under the stressful con-
ditions. It indicated that lignifications contributed to
the defence level of the plants to overcome the stress-
ful conditions via mediation of SM3. High PAL activi-
ties along with high lignifications in SM3-treated
plants clearly indicate activation of the phenylpropa-
noid pathway and a positive role of SM3 in the
process.
Activation of host defence response triggers the
complexity of the biochemical pathways within the
responding cell and new signal molecules are gener-
ated (Hammond-Kosack and Jones 1996), and it leads
to expression of defence genes. Defence genes are reg-
ulated by transcription factors and there are several
families of transcription factors which have significant
importance in plant stress responses. ERF transcrip-
tion factors play a vital role in both biotic as well as
abiotic stresses (Zhu et al. 2014). In some cases, it has
been found that the expression patterns of ERF and
other transcription genes are affected by priming with
rhizobacteria (Ballare 2014). Rhizobacteria-mediated
priming has already demonstrated (Van der Ent et al.
2009b) and such effect normally leads to activation of
JA- and ET-responsive genes (Van Wees et al. 1999;
Verhagan et al. 2004). Seed treatment with SM3
probably has also led to a priming-like effect in chick-
pea under the challenging conditions as evident by
activation of an ET responsive transcription factor
(ERF) CaTF2. ERF genes are present in different
legumes including chickpea, but their functions are
not well known. Higher expression of the ERF gene
J Phytopathol 163 (2015) 583–592 � 2014 Blackwell Verlag GmbH590
Actinomycete induce tolerance in chickpea S. Srivastava et al.
CaTF2 in the SM3-treated plants under the challenge
of both the pathogen and the NaCl reveals the
influence of the strain SM3 in activation of the ERF
transcription factor. Lower expression of the CaTF2 in
SM3 non-treated plants clearly confirms the role of
the stimulus generated by the strain SM3 in triggering
its expression.
Biocontrol potential of Streptomyces spp. was
revealed earlier (Yuan and Crawford 1995; Shekhar
et al. 2006). However, detail understanding of bio-
control mechanisms by Streptomyces spp. is still not
very clear. Similarly, the abiotic stress tolerance
mechanisms in plants following application of Strep-
tomyces spp. are also not very clear. The present
study thus not only reports the outcome of applica-
tion of SM3 in biotically and abiotically challenged
chickpea plants but also gives a meaningful insight
into some of the influences it had on the host while
alleviating the stresses. From the present study,
thus, it can be concluded that St. rochei SM3 proba-
bly has a priming-like effect on chickpea that was
mediated through ERF transcription factors resulting
in tolerance to challenges by both Sc. Sclerotiorum
and NaCl.
Conflict of Interest
The authors declare that there is no conflict of interest.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Different activities of Streptomyces spp.
J Phytopathol 163 (2015) 583–592 � 2014 Blackwell Verlag GmbH592
Actinomycete induce tolerance in chickpea S. Srivastava et al.
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