Streptomyces rochei SM3 Induces Stress Tolerance in Chickpea Against Sclerotinia sclerotiorum and...

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

J Phytopathol 163 (2015) 583–592 � 2014 Blackwell Verlag GmbH584

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


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



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


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



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.



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.



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.



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



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.



Streptomyces rochei SM3 Induces Stress Tolerance in Chickpea Against Sclerotinia sclerotiorum and...

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