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Evaluation of antagonistic and plant growth promoting activities of chitinolytic endophytic actinomycetes associated with medicinal plants against Sclerotium rolfsii in chickpea

May 2016
Journal of Applied Microbiology 121(2)

May 2016
121(2)

Authors:

Agricultural Research Organization ARO

Aims: To evaluate the potential of chitinolytic endophytic actinomycetes isolated from medicinal plants in order to diminish the collar rot infestation induced by Sclerotium rolfsii in chickpea. Methods and results: Sixty eight chitinolytic endophytic actinomycetes were recovered from various medicinal plants and evaluated for their chitinase activity. Among these isolates, twelve were screened for their plant growth promoting abilities and antagonistic potential against S. rolfsii. Further, these isolates were validated in vivo for their ability to protect chickpea against S. rolfsii infestation under greenhouse conditions. The isolates significantly (P < 0.05) increased the biomass (1.2-2.0 fold) and reduced plant mortality (42-75%) of chickpea. On the basis of 16S rDNA profiling, the selected antagonistic strains were identified as Streptomyces diastaticus, S. fradiae, S. olivochromogenes, S. collinus, S. ossamyceticus, and S. griseus. Conclusion: This study is the first report of the isolation of endophytic actinomycetes from various medicinal plants having antagonistic and plant growth promoting abilities. The isolated species showed potential for controlling collar rot disease on chickpea and could be useful in integrated control against diverse soil borne plant pathogens. Significance and impact of the study: Our investigation suggests that endophytic actinomycetes associated with medicinal plants can be used as bioinoculants for developing safe, efficacious, and environment-friendly biocontrol strategies in the near future. This article is protected by copyright. All rights reserved.

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ORIGINAL ARTICLE

Evaluation of antagonistic and plant growth promoting

activities of chitinolytic endophytic actinomycetes

associated with medicinal plants against Sclerotium rolfsii

in chickpea

S.P. Singh

and R. Gaur

1,2

1 Department of Microbiology, Mewar University, Gangrar, Chittorgarh, India

2 Department of Microbiology, Dr R. M. L. Avadh University, Faizabad, India

Keywords

Actinomycete, antifungal, chitinase, Cicer

arietinum L., endophyte, Sclerotium rolfsii.

Correspondence

Rajeev Gaur, Department of Microbiology,

Mewar University, Gangrar, Chittorgarh,

Rajasthan-312901, India.

E-mails: rajeevagaur@gmail.com;

singhsatyendra349@gmail.com

2016/0054: received 8 January 2016, revised

30 March 2016 and accepted 3 May 2016

doi:10.1111/jam.13176

Abstract

Aims: To evaluate the potential of chitinolytic endophytic Actinomycetes

isolated from medicinal plants in order to diminish the collar rot infestation

induced by Sclerotium rolfsii in chickpea.

Methods and Results: Sixty-eight chitinolytic endophytic Actinomycetes were

recovered from various medicinal plants and evaluated for their chitinase

activity. Among these isolates, 12 were screened for their plant growth

promoting abilities and antagonistic potential against Sc. rolfsii. Further, these

isolates were validated in vivo for their ability to protect chickpea against

Sc. rolfsii infestation under greenhouse conditions. The isolates signiﬁcantly

(P<005) increased the biomass (12–20 fold) and reduced plant mortality

(42–75%) of chickpea. On the basis of 16S rDNA proﬁling, the selected

antagonistic strains were identiﬁed as Streptomyces diastaticus,Streptomyces

fradiae,Streptomyces olivochromogenes,Streptomyces collinus,Streptomyces

ossamyceticus and Streptomyces griseus.

Conclusion: This study is the ﬁrst report of the isolation of endophytic

Actinomycetes from various medicinal plants having antagonistic and plant

growth promoting abilities. The isolated species showed potential for

controlling collar rot disease on chickpea and could be useful in integrated

control against diverse soil borne plant pathogens.

Signiﬁcance and Impact of the Study: Our investigation suggests that

endophytic Actinomycetes associated with medicinal plants can be used as

bioinoculants for developing safe, efﬁcacious and environment-friendly

biocontrol strategies in the near future.

Introduction

Actinomycetes, a group of Gram-positive ﬁlamentous

bacteria with high G +C ratio have been shown to be

attractive sources of natural compounds for the pharma-

ceutical and other industries. Recently endophytes have

attracted a lot of attention which is evident from the

increasing reports of beneﬁcial isolates from a wide range

of crops (Zhou et al. 2014; Golinska et al. 2015; Mingma

et al. 2015). Endophytic Actinomycetes have been

demonstrated to improve the plant growth as well as to

reduce infestation caused by phytopathogens through var-

ious mechanisms, including the production of bioactive

secondary metabolites, alteration in plant physiology, and

the stimulation of systemic acquired resistance in host

plants (Govindasamy et al. 2014). However, despite ever

increasing reports about endophytic microbes associated

with different plants, measures to understand their func-

tional role inside the host plant is limited. Most endo-

phytic Actinomycetes isolated to date generally belong to

the genus Streptomyces (Goodfellow and Simpson 1987)

which is responsible for the production of around 80%

Journal of Applied Microbiology ISSN 1364-5072

of the biologically active compounds. A recent report

suggested a possibility that Actinomycetes can protect

various plants by inhibiting the growth of potential

fungal pathogens via enzymes and antifungal compounds

(Goodfellow and Williams 1983). Among the various cat-

egories of enzymes, actinobacterial chitinases have been

widely demonstrated as inhibitors of fungal growth

(Taechowisan et al. 2003). Thus, because of the presence

of beneﬁcial traits, endophytic Actinomycetes have been

cited as promising biocontrol agents which act either

directly on fungal cell walls or are reported to initiate

increased plant responses against pathogen (Quecine

et al. 2008). Additionally, Actinomycetes are also reported

to promote plant growth by producing siderophores, sol-

ublization of insoluble phosphates, decomposition of

organic materials, such as cellulose, lignocellulose, starch

and chitin in soil as well as by production of growth pro-

moters such as indole-3-acetic acid (IAA) and gibberellic

acid (Taechowisan et al. 2005).

Sclerotium rolfsii is a plant pathogen known to cause

extensive damage to various agricultural and horticultural

crops (Harlton et al. 1995). Once the pathogen penetrates

the plant tissue, it produces a considerable mass of myce-

lium on the host surface. The disease symptoms, usually

undetectable, are dark-brown lesions on the root surface

and the ﬁrst visible symptoms are progressive yellowing

and wilting of leaves. This will eventually lead to the

plant death (Nene et al. 1991). The complete eradication

of Sc. rolfsii is nearly impossible due to the proliﬁc

growth and ability to produce large numbers of sclerotia

that persist in the soil for many years (Punja 1988). The

use of resistant cultivars and application of synthetic

chemicals are primary strategies for disease management,

but yield losses persist with numerous plants. The routine

usage of synthetic pesticides and fertilizers in agricultural

practices has drastically improved the crop yields, but on

the other hand has also caused an adverse effect on the

environment and human health. Thus, application of

chitinolytic endophytic Actinomycetes for inhibiting

Sc. rolfsii by these microbes could provide additional

opportunities for ecofriendly and sustainable disease

management. Efforts in this area by various researchers

have resulted in development of commercial biocontrol

preparations which have been reported to act against fun-

gal pathogens (Shimizu 2011). However, further studies

are needed to identify additional antagonistic Actino-

mycetes which can reduce disease caused by the notori-

ous phytopathogens including Sc. rolfsii. Considering this

aspect, application of chitinolytic endophytic Actino-

mycetes in agricultural might prove to be effective alter-

native against fungal pathogen, Sc. rolfsii.

Chickpea (Cicer arietinum L.) is one of the major food

legumes grown worldwide, and is an important

component of human and animal diet. The plant plays

an important role in maintenance of soil fertility in semi-

arid and arid zones of the world (Saxena 1990). However,

the successful cultivation of chickpea faces a serious

threat from the infestation of the fungal pathogen,

Sc. rolfsii (Nagamani et al. 2013; Sarkar et al. 2014). To

curb the losses, there is thus an urgency to develop/iso-

late endophytic Actinomycetes which can provide an

alternative for the successful management of Sc. rolfsii

infection in chickpea. Hence, the overall aims of this

investigation were to (i) selectively isolate chitinolytic

endophytic actinobacteria associated with the medicinal

plants, (ii) study their activity against Sc. rolfsii and other

fungal phytopathogens, (iii) determine their in vitro pro-

duction of active compounds related to plant growth

promotion and (iv) conduct an in vivo evaluation of the

efﬁcacy of potential antagonistic isolates as biological

control agents against Sc. rolfsii on chickpea.

Materials and methods

Sample collection

Healthy medicinal plants were collected from CSIR-

National Botanical Research Institute (Banthara) (26°550

N, 80°590E), Lucknow, India during June, 2013. The

selection of each plant for endophytic isolation was based

on its local ethnobotanical properties, including its

antibacterial, insecticidal, antitumor and wound-healing

and other medicinal properties (Jain 1994). Healthy root,

stem and leaf samples of each plant were placed in sterile

plastic bags and subjected to isolation procedures within

48 h. The representative plants selected for the study were

Rauvolﬁa serpentine,Gymnema sylvestre,Stevia crenata,

Bacopa monnieri,Andrographis paniculata and Withania

somnifera.

Enrichment and selective isolation of chitinolytic

endophytic Actinomycetes

Samples were air-dried for 6 h at room temperature and

then washed with an ultrasonic step (160 W, 15 min) to

remove the surface soils and adherent epiphytes com-

pletely. After drying, the samples were subjected to sur-

face sterilization procedure: 5 min wash in 4% sodium

hypochlorite (NaOCl), followed by 10 min wash in 2%

, a 5 min wash in 75% ethanol, a wash in sterile

water, and a ﬁnal rinse in 8% NaHCO

for 10 min. After

thoroughly drying under sterile conditions, the surface-

sterilized tissues were crushed in saline with autoclaved

mortar and pestle and subjected to suspensions contain-

ing 1% colloidal chitin in saline (085% w/v) in 1 : 10

ratio (Berger and Reynolds 1958). The suspensions were

S.P. Singh and R. Gaur Endophytic Actinomycetes against Sc. rolfsii

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incubated on a shaker at 28 2°C, 100 rev min

1

for

8–10 days for enrichment of chitinolytic Actinomycetes.

After incubation, 1 ml of six fold dilutions of individual

plant material suspensions were spread onto colloidal

chitin agar (CCA), consisting of the following con-

stituents (g l

1

): K

HPO

,07; KH

,03; NH

Cl, 10;

NaCl, 05; MgSO

5H

O, 05; FeSO

7H

O, 001; ZnSO

0001; MnCl

,0001; CaCl

2H

O, 0001; yeast extract,

005; CaCO

,0001; Bacto agar, 2% and colloidal chitin

1% (w/v) and incubated at 28 2°C for 1–2 weeks. Also

the noncrushed surface-sterilized tissues were dried in a

laminar airﬂow chamber and by using sterile scalpel,

outer tissues were removed. For the isolation of acti-

nobacteria, 05cm

of inner tissue size was carefully dis-

sected and placed directly over the surface of colloidal

chitin agar (CCA) media. The media was amended with

nystatin (50 lgml

1

) and cycloheximide (50 lgml

1

)

along with nalidixic acid (15 lgml

1

) and K

(01%) (to inhibit fast growing bacteria) in a ﬁnal con-

centration of 60 lgml

1

. The colonies appearing on the

plates were observed and selected carefully according to

their characteristics. After 10 days of incubation, the

colonies showing a clearing zone were selected as chiti-

nolytic Actinomycetes (Hoster et al. 2005). Actinobacte-

rial colonies growing out of the plated tissue segments

were transferred onto ISP2 media slants and repeatedly

subcultured until pure cultures were obtained.

Efﬁciency of surface sterilization

Two experiments were conducted to evaluate the efﬁcacy

of the surface sterilization procedures. Firstly, autoclaved

distilled water used in the last step of washing was used

to check the sterility of plant tissues (Schulz et al. 1993).

Secondly, surface-sterilized small segments (1–3 cm) of

plant tissues were also imprinted onto ISP 2 agar to con-

ﬁrm the effectiveness of the surface sterilization proce-

dures (Qin et al. 2009).

Quantiﬁcation of chitinase activity

Chitinase activity of isolates was quantiﬁed according to

the protocol of Gupta et al. (1995). The unit (U) activity

is deﬁned as the amount of enzyme releasing 1 lmol

N-acetylglucosamine (GlcNAc) per minute.

In vitro antagonistic bioassay

The isolates were evaluated for their biocontrol activity

towards four plant pathogenic fungi: Sclerotium rolfsii,

Rhizoctonia solani,Fusarium oxysporum and Alternaria

solani using dual culture in vitro assay (Singh et al.

2015b). The growth inhibition (%) was calculated by

comparing colony radius with the control plates. All the

experiments were conducted thrice with three replicates

each. The antagonistic activities of isolates were also

examined by scanning electron microscopy (SEM) of

7-day-old cultures grown on dual culture plates con-

tained potato dextrose agar (PDA) and glucose yeast malt

extract (GYM) agar (1 : 1).

Plant growth promoting activities

Phosphate solubilization activity of isolates was deter-

mined according to the methodology described by Mehta

and Nautiyal (2001). For quantitative estimation of

P-solubilization activity, isolates were used to estimate

the released phosphate concentration using the Fiske and

Subbarow method (1925). The phosphate solubilization

activity was expressed as equivalent phosphate (lgml

1

Catechol type and hydroxamate siderophores were esti-

mated by Arnow’s method (Arnow 1937) and Csaky test

(Csaky 1948) respectively. The amount of IAA produced

by Actinomycetes isolates was determined using GYM

broth supplemented with different concentration of

L-tryptophan (Basal, 0, 2 and 5 mg ml

1

) and incubated

at 30°C with shaking at 125 rev min

1

for 7 days

(Gordon and Weber 1951). Appearance of a pink colour

indicated IAA production. The level of IAA produced

was estimated by comparison with an IAA standard. Gib-

berellic acid (GA

) production was determined according

to Borrow et al. (1955). The absorbance was recorded at

254 nm using UV spectrophotometer and calibrated the

amount of GA in lgml

1

by comparison with a stan-

dard of gibberellic acid solutions of known concentration.

All the in vitro experiments were performed thrice with

three replicates.

In vivo biocontrol and plant growth promotion assay

Preparation of microbial inoculums

The pure Actinomycetes isolates were individually inocu-

lated in GYM broth and incubated at 30 2°C for

7–10 days under shaking conditions. The culture was cen-

trifuged at 8000 gfor 5 min. The cell pellet was suspended

in 085% saline and the cell density was adjusted to

10910

CFU ml

1

using spectrophotometer at 610 nm

(Singh et al. 2015a). The pathogen Sc. rolfsii was isolated

by collecting sclerotia produced on infected collar region

of chickpea plants. Surface sterilization of sclerotia was

done by dipping them in 1% (v/v) NaOCl for 5 s followed

by washing thrice with sterilized distilled water. The sclero-

tia were then placed onto PDA plates and incubated at

28°C for 7 days. The cultures were puriﬁed by picking a

single sclerotium and transferred into a fresh PDA plate.

Pathogen inoculum was prepared by growing Sc. rolfsii in

Endophytic Actinomycetes against Sc. rolfsii S.P. Singh and R. Gaur

sterile cornmeal sand (240 g of clean quartz sand, 60gof

yellow cornmeal, and 75 ml of SDW; moisture level 50%)

for 2 weeks at 25°C in the dark with proper mixing at reg-

ular intervals for uniform distribution of fungal mycelia

(Abeygunawardena and Wood 1957).

Seed priming with endophytic Actinomycetes isolates

The surface sterilization of chickpea (cv. Radhey) seeds

was carried out with 2% NaOCl solution for 1 min, fol-

lowed by washing with sterile water to remove traces of

NaOCl. Plastic pots (15 910 cm) were used for sterile

soil assay and 15 kg of the potting mixture was ﬁlled in

each pot. For seed treatment, chickpea seeds were coated

with Actinomycetes isolates cell suspensions prepared in

10% (w/v) carboxyl methyl cellulose, CMC (HiMedia,

Mumbai, India) which was used as an adhesive. The

coated seeds were air-dried under a stream of sterile air

for 2–3 h. The coated seeds were then planted in pots

with an average of four germinated seeds per pot at a

depth of approx. 15 cm. The following treatments were

examined: (i) Control: neither inoculated with pathogen

(Sc. rolfsii) nor with biocontrol isolate; (ii) Con-

trol +Sc. rolfsii: inoculated with pathogen (Sc. rolfsii) but

not with biocontrol isolate; (iii-xiv) pathogen (Sc. rolf-

sii)+biocontrol agents (CC1, CC4, CC12, CC20, CC23,

CC29, CC31, CC38, CC41, CC42, CC52 and CC53). The

inoculum of Sc. rolfsii was applied in the soil of the pots

adhering in to the plants in all treatments at the rate of

50 g per pot after plants were of 3 weeks old. The experi-

ment was conducted in a greenhouse in controlled condi-

tions of 10 h dark, 14 h light and temperature of

23 2°C at experimental area of CSIR-NBRI from

November to January 2013–2014. The pots were arranged

in a complete randomized design (CRD) in a set of seven

replicates for each treatment and the experiment was

repeated twice. Data from the repeated experiments were

pooled for analysis. After 3 weeks of pathogen inocula-

tion the experiment was terminated to record yield

related attributes viz. plant weight, fruiting, ﬂowering,

nodulation and plant mortality.

In vivo gas exchange measurements

The gas exchange parameters of net photosynthesis rate,

stomatal conductance and transpiration rate were moni-

tored in fully expanded chickpea leaves at ﬂowering stage

with Li-Cor 6400 gas exchange portable photosynthesis

system (Li-Cor, Lincoln, NE) (Singh et al. 2014).

Molecular identiﬁcation and presence of biosynthetic

genes

The isolates were subjected to 16S rRNA gene sequence

analysis for precise genus and species identiﬁcation. The

16S rRNA genes from pure cultures were ampliﬁed using

the universal primer pair (Qin et al. 2009). The PCR

products were separated by agarose gel electrophoresis,

puriﬁed using QIAquick gel extraction kits (Qiagen, Hil-

den, Germany), and sequenced on an ABI Prism 3730

sequencer. The 16S rRNA gene sequences determined was

compared with the GenBank/EMBL/DDBJ databases

using the BLASTN search program. A phylogenetic tree was

constructed by the neighbour-joining method using the

MEGA 5 software package, after pairwise alignments using

the CLUSTAL X 1.8 program (Tamura et al. 2011). The sta-

bility of relationships was assessed by performing boot-

strap analyses of the neighbour-joining data based on

1500 re-samplings. Detection of biosynthetic genes was

also done using three sets of degenerate primers for the

ampliﬁcation of genes encoding polyketide synthases I

and II (PKS I and PKS II) and nonribosomal peptide

synthetases (NRPS) from the isolates tested positive for

biocontrol ability according to Qin et al. (2009).

Data analysis

To study the level of signiﬁcance, analysis of variance

(ANOVA) using SPSS package (SPSS ver. 16.0, SPSS Inc., Chi-

cago, IL) for randomized complete block design. Signiﬁ-

cant differences among treatments were based on the

Tukey’s multiple range test at P<005.

Results

Isolation and screening of endophytic Actinomycetes for

characteristic traits

A total of 68 Actinomycetes isolates were isolated from

endophytic regions of healthy medicinal plants (Fig. S1).

Of the 68 isolates, the majority (n=27, 3970%) were

isolated from roots followed by leaf (n=24, 3529%),

and stem (n=17, 25%) (Table S1). Among these iso-

lates, only 12 isolates showed better chitinolytic poten-

tials, depicting more than 25 mm clearing zone (Hi-

antibiotic zone scale, HiMedia). The isolates depicting

chitinase activity were further validated by quantiﬁcation

of chitinase enzyme. An ascending trend in enzyme pro-

duction was recorded between 2nd to 6th day of incuba-

tion (Table S2), after which a gradual decline in the

enzyme activity was observed in most of the isolates. The

results indicated a positive correlation between growth

and enzyme activity. The 12 isolates exhibited potent

chitinase activity (047–109 U ml

1

) with CC53 showing

the highest chitinase activity (109 002 U ml

1

) fol-

lowed by CC4 (096 001 U ml

1

) at 6th day of incu-

bation. All these isolates were therefore selected for

antagonistic activity against four major fungal pathogens.

S.P. Singh and R. Gaur Endophytic Actinomycetes against Sc. rolfsii

Test for antagonism

Twelve Actinomycetes isolates namely CC1, CC4, CC12,

CC20, CC23, CC29, CC31, CC38, CC41, CC42, CC52

and CC53 revealed a completely antagonistic nature

against all the four fungal pathogens viz., Sc. rolfsii,

R. solani,F. oxysporum,A. solani after 5 days of incuba-

tion. Among the positive isolates, the greatest inhibition

of fungal colony growth was noticed with CC53 (703%),

CC38 (692%) and CC52 (678%) which was more than

60% against Sc. rolfsii. The isolates CC4 (615 and

664%) and CC53 (630 and 676%) showed maximum

inhibition against R. solani and F. oxysporum respectively.

The isolates CC38, CC41 and CC53 showed >600% inhi-

bition against A. solani (Table S3). The biocontrol activ-

ity against all the four fungal pathogens was exhibited by

isolates CC23 and CC53 (Fig. 1). Among these four iso-

lates, the maximum antagonistic activity was shown by

CC53. Moreover, the isolate CC53 inhibited Sc. rolfsii

growth up to 703% in the dual culture plate assay

(Table S3; Fig. 1b1). The mycoparasitic activity of CC53

was conﬁrmed by the SEM observation at the interaction

zone of CC53 and Sc. rolfsii (Fig. 2a–f). Microscopic

observations revealed complete destruction of Sc. rolfsii

mycelia by the coiling and spore proliferation of CC53

(Fig. 2d–f).

Plant growth promoting traits of endophytic

Actinomycetes

The isolates were screened for plant growth promoting

traits and our results indicated that all the 12 isolates were

able to solubilize phosphate and produced siderophore,

IAA and GA

. Phosphate solublization ranged from 339

to 663lgml

1

at 7 days after inoculation. The maxi-

mum phosphate solubilization activity was found in CC53

(663lgml

1

) followed by CC4 (654lgml

1

) and

CC42 (618lgml

1

) (Table S4). These isolates were also

analysed for siderophore production. Siderophore pro-

duction ranged from 67to279lgml

1

(for catechols)

and 120–971lgml

1

(for hydroxymate) at the 7th day

after inoculation (Table S4). The greatest levels of hydrox-

amate type siderophore were produced by CC53

(5358 lgml

1

) and CC4 (4187 lgml

1

). Table S4 also

depicts the other plant growth promoting activities of the

selected 12 isolates such as IAA and GA

production. Fur-

thermore, these isolates were screened for their ability to

produce IAA, with different concentrations of tryptophan

(0, 2 and 5 mg l

1

). The IAA production without trypto-

phan was in the range of 32–159lgml

1

. The incre-

ment in IAA production was observed with the increasing

concentration of tryptophan i.e. for 2 mg l

1

tryptophan,

the concentration ranged from 117to394lgml

1

and

for 5 mg l

1

the concentration ranged from 201to

646lgml

1

(Table S4). Three isolates (CC53, CC4 and

CC42) showed more than 60 lgml

1

IAA production

while least activity was found with CC31 (202lgml

1

)

in 5 mg l

1

of tryptophan concentration. Similarly, the

production of GA

ranged from 30to813 lgml

1

in all

the 12 isolates at the 7th day after inoculation (Table S4).

Total Biomass of chickpea after endophytic

Actinomycetes priming

We further analysed the potential of our isolates to see

their effect on biomass production in control

(untreated uninoculated control), control +pathogen

Sc. rolfsii (untreated pathogen inoculated control) as

well as pathogen +biocontrol agents (Actinomycetes

primed inoculated with pathogen) treatments. All the

endophytic Actinomycete isolates showed signiﬁcant dif-

ference (P<005) in terms of plant growth parameters

compared to the untreated Sc. rolfsii inoculated and

control plants (Fig. 3). The growth parameters inclusive

of fresh and dry biomass yield were signiﬁcantly

(P<005) enhanced in all the Actinomycetes treat-

ments as compared to the control and con-

trol +Sc. rolfsii (Table 1). The maximum increase in

shoot and root length by 18 and 22 fold, respectively,

was observed in CC53 treated plants followed by CC42

(17 and 19 fold respectively) (Table S5). However, the

degree of growth promotion in terms of ﬂowering

(15–25 fold), branches (12–22 fold) and nodulation

(12–22 fold) varied with various endophytic Actino-

mycete treatments (Table S5). Among the treatments,

maximum plant biomass was achieved with CC53 and

CC23 which showed increments of 19 and 20 fold

followed by CC42 with a 17 and 19 fold enhancement

in fresh and dry biomass respectively when compared

with the Sc. rolfsii inoculated control (Table 1). The

maximum number of fruiting bodies were also observed

in treatments with CC53 and CC4 (23 fold) followed

by CC23 (22 fold) and CC38 (20 fold) with respect

to Sc. rolfsii inoculated control. After 3 weeks of

Sc. rolfsii inoculation, symptoms developed in con-

trol +Sc. rolfsii plants. The plants treated with endo-

phytic Actinomycetes showed their efﬁcacy against

Sc. rolfsii by drastically reducing the plant mortality

3 weeks after inoculation (Table 1). The most efﬁcient

reduction of Sc. rolfsii infection was shown by CC53

which signiﬁcantly (P<005) reduced the plant mortal-

ity (25%), followed by CC23 and CC42 which demon-

strated 31% against the control +Sc. rolfsii (Table 1).

The maximum mortality (more than 60%) was

observed in CC29, CC1 and CC4 treatments. A signiﬁ-

cant increase in colonization was observed in plants

Endophytic Actinomycetes against Sc. rolfsii S.P. Singh and R. Gaur

treated with endophytic Actinomycetes CC53, CC23

and CC42 followed by CC4 and CC38 (Table 1).

In vivo gas exchange measurements

We further estimated the gas exchange parameters in

control +pathogen and biocontrol treated chickpea

leaves. Our data showed marked reduced net photosyn-

thetic rate in chickpea leaves in pathogen inoculated leaf,

whereas application of endophytic Actinomycetes

increased the photosynthetic rate from 14to21 fold

(Fig. S2). Similarly, the transpiration rate (11–21 fold)

and stomatal conductance (13–22 fold) were signiﬁ-

cantly increased in endophytic Actinomycetes treated

(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

CC23

CC 53

Streptomyces

olivochromogenes

Streptomyces griseus

S.rolfsii

R.solani

F.oxysporum F.oxysporum

A. solani A. solani

R.solani

S.rolfsii

Figure 1 In vitro evaluation of antifungal activity. Chitinolytic Actinomycetes were tested in a dual culture assay against pathogenic fungi; Strep-

tomyces olivochromogenes and Streptomyces griseus against (a1; b1) Sclerotium rolfsii, (a2; b2) Rhizoctonia solani, (a3; b3) Fusarium oxysporum,

and (a4; b4) Alternaria solani.

S.P. Singh and R. Gaur Endophytic Actinomycetes against Sc. rolfsii

chickpea plants with respect to control + Sc. rolfsii plants

(Fig. S2).

Molecular identiﬁcation and detection of biosynthetic

genes

On the basis of their ability to colonize chickpea roots

and promote plant growth under greenhouse conditions,

six potent Actinomycete isolates were selected for

molecular identiﬁcation. PCR ampliﬁcation was done

with speciﬁc primer for the 16S-rRNA region generated

bands ranging from 1450 to 1500 bp. These fragments

were sequenced separately, and the sequence data were

deposited in the GenBank of the National Center for

Biotechnology Information (NCBI) with accession num-

ber KT962908-13. Homology search and Blast analysis

revealed that the isolated strains CC4,CC20, CC23,

CC38, CC42 and CC53 belonged to genus Streptomyces

(a) (b) (c)

(d) (e) (f)

S.rolfsii

S.rolfsii S.rolfsii

S.rolfsii

S.rolfii

CC53

Streptomyces

griseus

CC53

7/8/2014 HV HFW mag

10 000 ×

dwell

10·00 kV14·9 µm

5 µm

label3 µs5:34:38 PM

7/8/2014 HV HFW mag

5 000 ×

dwell

10·00 kV 29·8 µm

10 µm

label3 µs5:35:52 PM

7/8/2014 HV HFW mag

2 000 ×

dwell

10·00 kV 74·6 µm

30 µm

label3 µs5:36:18 PM

7/7/2014 HV HFW mag

10 000 ×

dwell

10·00 kV14·9 µm

5 µm

label3 µs

5:02:34 PM

7/8/2014 HV HFW mag

5 000 ×

dwell

10·00 kV 29·8 µm

10 µm

label3 µs

5:16:56 PM

7/8/2014 HV HFW mag

2 000 ×

dwell

10·00 kV 74·6 µm

30 µm

label

3 µs5:17:44 PM

Figure 2 Scanning electron micrographs of isolate CC53 (S.s griseus SP12); (a, b, c) mycelia structure of Sclerotium rolfsii; (d, e) arrows showing

mycoparasitic activity of CC53 against Sc. rolfsii in dual culture assay and (e) complete destruction of Sc. rolfsii mycelia.

Control Control

+S. rolfsii CC1 CC4 CC12 CC20 CC23 CC29 CC31 CC38 CC41 CC42 CC52 CC53

Figure 3 Effect of various endophytic Actinomycetes with respect to control on growth of chickpea.

Endophytic Actinomycetes against Sc. rolfsii S.P. Singh and R. Gaur

diastaticus SP2, Streptomyces fradiae SP4, Streptomyces

olivochromogenes SP5, Streptomyces collinus SP8, Strepto-

myces ossamyceticus SP10 and Streptomyces griseus SP12

respectively. Phylogenetic analyses of the isolates based

on 16S- rRNA gene sequence has been shown in Fig. 4.

We further investigated the potential of endophytic

Actinomycetes to synthesize bioactive compounds, as

indicated by the presence of NRPS and PKS genes. The

presence of the genes encoding PKS I, PKS II and NRPS

were detected in six potent Actinomycetes using three

sets of degenerate primers. Only strain, S. griseus SP12

showed positive ampliﬁcation products with the PKS I,

PKS II and NRPS primers (Table S6). Streptomyces

diastaticus SP2, S. fradiae SP4, S. collinus SP8 and S. os-

samyceticus SP10 showed positive ampliﬁcation products

with the PKSI and NRPS primers while S. olivochromoge-

nes SP5 showed only positive ampliﬁcation products with

NRPS (Table S6).

Discussion

Endophytic micro-organisms can play a signiﬁcant role as

biocontrol agents and are considered to beneﬁcial for

plant disease management (Shimizu 2011). It has been

observed that among the various microbial inoculants,

endophytic micro-organisms are the most promising

group for their beneﬁcial exploitation in the ﬁeld of agri-

culture and pharmaceutics (Kunoh 2002). However, in-

spite of the signiﬁcant work done earlier, there is still a

paucity of knowledge on the actual endohpytic Actino-

mycetes possessing biocontrol activity. Endophytic Acti-

nomycetes from different medicinal plants are reported as

major source of natural products with potential

antifungal activity (Golinska et al. 2015; Passari et al.

2015). These ﬁndings encouraged us to explore tradi-

tional medicinal plants for understanding the endophytic

Actinomycetes community and their potential as biocon-

trol agents and plant growth promoters. In this study, we

isolated 68 different chitinolytic endophytic Actinomycete

strains from different parts of the medicinal plants using

enrichment method. The maximum numbers of Actino-

mycete isolates were recovered from R. serpentina. The

isolation of endophytic actinobacteria is a critically

important procedure in the present study. Although Acti-

nomycetes were isolated from different tissues, isolate

ratios were highest in root tissues indicating that endo-

phytic Actinomycetes are most dominant in the root

parts. Our results are consistent with the ﬁndings of

Taechowisan et al. (2003) and Verma et al. (2009) who

stated that roots represent a good territory for endophytic

Actinomycetes. This may be owing to the fact that soil

Actinomycetes can move along the plant roots very easily.

Also, Actinomycetes can colonize roots by entering via

cracks at the lateral root junctions (Coombs and Franco

2003).

Importance of chitinolytic endophytic Actinomycetes

in control of disease caused by fungal pathogens has also

been recognized. Besides many factors, chitinase enzyme

action on fungal pathogen cell wall seems to play an

active role in antagonism (Nagpure et al. 2014). Out of

68 strains, only 12 strains exhibited strong chitinolytic

activity as determined by degradation of colloidal chitin

on CCA plates. In the present investigation, the isolates

showed signiﬁcant antifungal activity against all the four

pathogens, that is, Sc. rolfsii,R. solani,F. oxysporum and

A. solani. Possibility exists that apart from the production

Table 1 Effect of endophytic isolates in chickpea for promotion of plant health parameters and plant mortality inoculated with Sclerotium rolfsii

Treatments Fresh Weight (g pot

1

) Dry Weight (g pot

1

) Fruiting (no.) Plant mortality (%) CFU (log

CFU g

1

)

Control 2346 089

efg

569 055

abc

1600 089

000 000

–

Control +Sc. rolfsi 1878 213

419 086

1400 155

9000 288

–

CC1 2799 134

699 069

abc

1600 196

6400 346

161 008

CC4 3094 140

774 079

3183 198

3800 231

336 0013

CC12 2147 128

fgh

537 076

1700 146

5400 231

265 026

abc

CC20 2852 113

713 073

2700 178

3800 173

266 015

abc

CC23 3567 151

825 160

3000 229

3100 231

341 054

CC29 2470 124

617 074

abc

2400 175

6800 173

167 027

CC31 2375 122

677 103

abc

2367 214

5100 058

148 005

CC38 2944 119

736 075

2867 214

3400 231

296 030

abc

CC41 2094 123

590 076

abc

1633 059

6100 173

205 021

cde

CC42 3247 119

811 173

2700 096

3100 173

338 025

CC52 3043 114

bcd

761 103

1967 139

4100 115

257 044

bcd

CC53 3634 227

857 115

3217 165

2500 288

352 058

Values are mean of six replicates with standard error (SE) are indicated. Means followed by the same letter(s) within the column are not

signiﬁcantly different according to Tukey’s multiple comparison test (P<005).

S.P. Singh and R. Gaur Endophytic Actinomycetes against Sc. rolfsii

and excretion of chitinolytic enzymes production of sec-

ondary metabolites might have played a key role in the

antagonistic action of the positive Actinomycete isolates.

The ﬁndings of our study are parallel to some of the pre-

vious studies where endophytic Actinomycetes are

reported as potential antifungal agents (Strobel 2003;

Verma et al. 2009; Shimizu 2011). Further as depicted in

our study, antagonistic behaviours of endophytic Actino-

mycetes against fungal pathogens through several mecha-

nisms such as mycoparasitism have been explained using

scanning electron microscopic studies. As evident from

the SEM images, we can interpretate the S. griseus SP12

intertwines around the fungal pathogen mycelia and per-

forates it, leading to its death due to cytoplasmic extru-

sion. Similar results were observed with chitinolytic

Actinomycete Streptomyces toxytricini against R. solani

(Patil et al. 2010) under SEM observation with the excep-

tion of hyperparasitism of antagonistic isolates.

All these isolates were found positive for phosphate

solubilization, production of siderophores and

Streptomyces diastaticus JX524717

100

Streptomyces diastaticus strain SP2 KT962908

Streptomyces fradiae JF682780

Streptomyces coelicoflavus KJ573027

Streptomyces ambofaciens FJ486314

Streptomyces lienomycini NR 112464

Streptomyces tendae KF454844

Streptomyces collinus subsp. albescens KC119160

Streptomyces griseorubens EU570500

Streptomyces griseus AB030571

Streptomyces griseus strain SP12 KT962913

Streptomyces griseus subsp. griseus NR 074787

Streptomyces caviscabies NR 114493

Streptomyces globisporus NR 044145

Streptomyces albiflavescens KC771426

Streptomyces olivochromogenes EU589442

Streptomyces olivochromogenes strain SP5 KT962910

Streptomyces neyagawaenis AB026219

Streptomyces torulosus NR112472

Streptomyces sp. EF608465

Streptomyces hygroscopicus subsp. ossamyceticus AY999876

Streptomyces ossamyceticus strain SP10 KT962912

Streptomyces flavoviridis EU570674

Streptomyces collinus strain SP8 KT962911

Streptomyces avidinii FJ481066

Streptomycetes aurantiogriseus NR 115385

Streptomyces fradiae strain SP4 KT962909

Actinomycetales bacterium AY944250

Figure 4 Neighbour-joining phylogenetic tree analysis of 16S rRNA of endophytic Actinomycetes. The numbers at the nodes indicate the levels

of bootstrap support (%) based on 1500 re-sampled data sets. The scale bar corresponds to 002 substitutions per nucleotide position. Numbers

in parentheses indicate accession numbers in Genbank.

Endophytic Actinomycetes against Sc. rolfsii S.P. Singh and R. Gaur

phytohormones (IAA and GA

). The result obtained by

this study is consistent with our ﬁndings in which all iso-

lates were identiﬁed as phosphate solubilizers. This may

be either due to the acidiﬁcation of medium by produc-

tion of low molecular weight organic acids (Hamdali

et al. 2008). The endophytic Actinomycetes present inside

plant tissues are known to produce growth hormones

that may play an important role in plant development.

Among the various kinds of growth promoters, Actino-

mycetes are well known for the production of hydroxam-

ate and catecholate type siderophores, which can inhibit

the growth of the phytopathogen by competing for iron

in the environment (Khamna et al. 2009). Singh et al.

2015b suggested that the production of siderophore is an

important factor for phytopathogen antagonism and

growth of the plant. Similarly, Verma et al. (2009) also

showed that endophytic Actinomycetes isolated from

Azadirachta indica exhibited high amount of siderophore

and IAA production. It is also suggested that phosphate

solubilization, production of phytohormones, and other

related compounds by the Actinomycetes will interact

with the plants as part of its colonization, leading to

growth promotion, induced resistance, and modulation

of plant defence mechanisms (Goudjal et al. 2014; Singh

et al. 2015a,b).

In our study, the selected active endophytic Actino-

mycetes demonstrated a signiﬁcant role in plant growth

activity and reduced in vivo collar rot of chickpea caused

by Sc. rolfsii plant pathogen under greenhouse experi-

ment. Endophytic Actinomycetes treated plants displayed

maximum increase in physiological parameters such as

plant biomass, plant length, fruiting, ﬂowering, branches

and nodulation of the chickpea plant. The results indi-

cated that the plant growth related activities and antago-

nism is likely to be a major mechanism employed by

these Actinomycetes to control Sc. rolfsii in the chickpea.

The production of antibiotics by Actinomycetes may play

an important role in the biocontrol of pathogens and

contribute to the enhancement of plant growth (Pala-

niyandi et al. 2013; Goudjal et al. 2014). The isolate

S. griseus SP12 (CC53), in addition to its capacity to

reduce the disease severity of Sc. rolfsii, also increased

the total biomass of the chickpea. The gas exchange

parameters such as net photosynthesis, stomatal conduc-

tance and transpiration rate were also signiﬁcantly

increased in endophytic Actinomycetes treated chickpea

plants. Many reports have shown that endophytic Acti-

nomycetes can increase growth in different crops (Misk

and Franco 2011; Bhattacharyya and Jha 2012), such an

increase may confer advantages to the host plant with

respect to health and overall plant development. The

ﬁndings are in line with the previous studies (El-Tarabily

et al. 2009; Pattanapipitpaisal and Kamlandharn 2012)

where the authors demonstrated that application of chiti-

nolytic endophytic Actinomycetes in plant seedlings pro-

duced a signiﬁcant increase in biomass along with the

disease reduction. It can also be hypothesized that the

application of endophytic Actinomycetes might have per-

formed as a strong sensor for the generation of phyto-

defence responses against fungal phytopathogens (Kunoh

2002).

In soil systems, a positive correlation was observed

between degradation of chitin hydrolysis by chitinolytic

microbes and microbial abundance (Quecine et al. 2008;

Kielak et al. 2013). In the present study, root colonization

showed a signiﬁcant growth in Actinomycetes treated

plants, possibly because of higher chitin hydrolysis and

antagonism activity. There is also added possibility that

the chitinolytic endophytic Actinomycetes might have

actually multiplied to a sufﬁcient extent under the experi-

mental conditions because of the presence of fungal myce-

lium which is utilized as a rich source of chitin. Also, role

of chitinolytic Actinomycetes cannot be ignored as our

assumption is in agreement with the ﬁndings of the earlier

research, which have also showed positive relationship

between microbial diversity and chitinase activity (Beier

and Bertilsson 2013). Furthermore, viability and coloniza-

tion of the chitinolytic Actinomycetes in soil was

enhanced, which might have provided persistent protec-

tion to the plants from Sc. rolfsii.

In this study, diverse group of endophytic Actino-

mycetes belonging to genus S. diastaticus SP2, S. fradiae

SP4, S. olivochromogenes SP5, S. collinus SP8, S. os-

samyceticus SP10 and S. griseus SP12 were identiﬁed as

potent chitinase producers. This is the ﬁrst report of

S. diastaticus SP2, S. fradiae SP4, S. olivochromogenes SP5,

S. collinus SP8, S. ossamyceticus SP10 and S. griseus SP12

isolated as endophytes from the medicinal plants namely

B. monnieri,W. somnifera, A. paniculata, Stevia crenata,

G. sylvestre and R. serpentine respectively.

To test the biosynthetic potential of these isolates,

presence of genes encoding PKS and NRPS, responsible

for the synthesis of most biologically active compounds

in Actinomycetes was investigated (Qin et al. 2009;

Passari et al. 2015). However, the true antifungal activity

of the endophytic Actinomycetes may only be assessed by

screening of biocontrol potential against desired patho-

gens. In this study, most of the isolates showed the pres-

ence of genes that encoded PKS I, PKS II and NRPS

enzymes, which also showed antifungal activity against

most of the tested phytopathogens. However, ﬁve isolates

(S. diastaticus SP2, S. fradiae SP4, S. olivochromogenes

SP5, S. collinus SP8 and S. ossamyceticus SP10) that were

positive for PKS I and NPRS but negative for PKS II, also

demonstrated signiﬁcant antifungal potential. Only one

of the isolates (S. griseus SP12) possesaaaased all three

S.P. Singh and R. Gaur Endophytic Actinomycetes against Sc. rolfsii

genes (PKS I, PKS II and NRPS). This isolate showed

maximum antifungal activity. These results indicate that

the isolates possessed biosynthetic genes that are associ-

ated with antagonism against the pathogens tested. Lack

of ampliﬁcation of PKS I and PKS II in some of the iso-

lates may be due to absence of these genes (Ayuso-Sacido

and Genilloud 2005). Moreover, the NRPS genes involved

in the secondary metabolites production are known to

play a role in antagonistic behaviour of the micro-organ-

ism possessing them. The results in the present study are

in agreement with a previous study (Ayuso-Sacido and

Genilloud 2005) where the authors illustrated that pres-

ence of functional genes in endophytic Actinomycetes

was associated with antagonistic behaviour. The pre-

screening of isolates with degenerate primers targeting

functional genes of bioactive compounds is thus an effec-

tive approach for detecting novel and useful secondary

metabolites (Weber et al. 2015).

The current investigation thus indicates that microbial

residents of the plant tissues represent a potential reser-

voir of antagonism that is capable of challenging the fun-

gal pathogens. These strains showed maximum

production of chitinase with effective suppression of phy-

topathogens. To our knowledge, this is the ﬁrst study

where endophytic actinobacteria were successfully isolated

on the basis of chitinase activity from different medicinal

plants. These species also enhanced the growth of plants

by solubilizing inorganic phosphorous and producing

siderophores and phytohormones (IAA; GA

). The plant

growth promoting traits of isolated Actinomycetes and its

biocontrol activity against Sc. rolfsii can be further uti-

lized for increasing the yield abilities in chickpea. These

ﬁndings provide persuasive evidence that the chitinolytic

endophytic Actinomycetes residing in the healthy plants

tissues offer options for sustainable agriculture. Develop-

ment of such prospective and viable technology is thus

the need of the hour which can not only act as a key for

better agronomic cultivations but also be useful for

improved biomass and better management of the fungal

pathogens. The study also suggests that augmenting these

populations of crops would make a great addition of an

integrated management plan that employs server disease

management strategies increasing the sustainability of the

crop production in the near future.

Acknowledgements

The authors are grateful to the Director, CSIR-National

Botanical Research Institute, Lucknow, India, for provid-

ing ﬁnancial support and necessary facilities during the

course of investigation. Help extended by Dr. Aradhana

Mishra for encouragement is greatly appreciated.

Conﬂict of Interest

The authors declare that there exists no potential conﬂict

of interest among them.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Figure S1 Selective isolation of chitinolytic endophytic

Actinomycetes.

Figure S2 Effect of endophytic Actinomycetes on net

photosynthesis (a) stomatal conductance, (b) transpira-

tion rate and (c) of chickpea.

Table S1 Percentage recovery of endophytic Actino-

mycetes isolated from different tissues of medicinal

plants.

Table S2 Quantiﬁcation of chitinolytic activity

(U ml

1

) of selected endophytic Actinomycetes isolates.

Table S3 In vitro screening of endophytic Actino-

mycetes for antifungal activity (diameter of inhibition

zone %).

Table S4 In vitro screening of endophytic Actino-

mycetes for beneﬁcial characteristic traits.

Table S5 Effect of endophytic Actinomycetes in chick-

pea for promotion of plant growth parameters inoculated

with Sclerotium rolfsii.

Table S6 Presence of polyketide synthases (PKS I and

II) and nonribosomal peptide synthetases (NRPS) genes

of endophytic Actinomycetes isolated from medicinal

plants.

Endophytic Actinomycetes against Sc. rolfsii S.P. Singh and R. Gaur

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An actinomycetes isolate of Loktak Lake soil, designated as MT7, was characterized and identified as Streptomyces sp. based on fatty acid methyl ester and 16S ribosomal RNA gene analysis. Streptomyces sp. MT7 showed strong and broad spectrum antagonism towards seven out of eight tested wood-rotting fungi. Strain MT7 secretes three vital fungal cell wall lytic enzymes, i.e. chitinase, β-1,3-glucanase, and protease, and siderophores. Extracellularly produced mycolytic enzymes lost their antifungal activity completely after treatment with proteinase K and heat, indicating that the tested antifungal metabolites are heat-sensitive and proteinaceous in nature. Extracellular fluid (ECF) and its organic solvent extract also exhibited potential antagonism towards the tested wood-rotting fungi. Antifungal metabolites were characterized as polyene in nature. Biocontrol traits like co-production of cell wall lytic enzymes and antifungal secondary metabolites including siderophores by Streptomyces sp. MT7 suggests that it could be employed as a potential biocontrol agent against wood-rotting basidiomycetes.

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The ability of Streptomyces species to act as biocontrol agents for plant pathogens via induced systemic resistance has been demonstrated and considerable efforts have been made in elucidating the underlying mechanisms of Streptomyces-host plant-phytopathogen interactions. Here, we have assessed the ability of Streptomyces coelicolor, Streptomyces griseus, Streptomyces albus, Streptomyces antibioticus and Streptomyces champavatii to provide disease protection against Rhizoctonia solani in Solanum lycopersicon and have also examined associated changes in hydrogen peroxide (H2O2) production, lipid peroxidation (LPO) and antioxidant enzymes. The production of H2O2 at the second day after pathogen inoculation (dapi) was observed to be 1.1-fold higher in Streptomyces-treated plants, when compared to untreated inoculated control plants. A similar increase in catalase and ascorbate peroxidase activity was observed at fourth dapi whereas increased activities of guaiacol reductase and glutathione peroxidase were observed at fifth dapi. Likewise, LPO reached a maximum at sixth dapi in untreated inoculated plants while in Streptomyces-treated plants it was observed to be 1.3-1.5-fold less when compared to untreated inoculated control plants. This study offers novel insights into the mechanisms of priming by Streptomyces and highlights their capacity to activate plant defence responses generated by biotic stress through induction of antioxidant enzymes along with improved reactive oxygen species management.

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Sep 2012

Two hundred and eighty three strains were isolated from rhizoshere-associated soils, from Ubon Ratchathani and Srisaket province, using Enrichment Media for isolation of Chitinase-producing Actinomycetes agar (EMCA agar). All strains were screened for chitinolytic activity and sixty eight strains gave significant clear zone on EMCA agar plates. The selected chitinolytic strains were assayed for in vitro antagonism against Sclerotium rolfsii using cornmeal agar (CMA agar) assay procedure and the result showed that thirteen isolates have remarkable inhibiting the growth of the fungus and the top five antagonistic actinomycetes were PACCH 277, PACCH129, PACCH225, PACCH24 and PACCH246, respectively. The result indicated that these actinomycetes produce chitinase which catalyze the degradation of chitin, resulting in inhibition of S. rolfsii growth. Their abilities to control the disease development were tested for in vivo biocontrol assay on chilli seedlings. Two out of thirteen candidate, PACCH24 and PACCH225, antagonists reduced the disease development at 90%. It was suggested that the ability to inhibit the growth of pathogen in vitro was not related to the disease reduction in vivo. The strain PACCH24 was further identified as Streptomyces hygroscopicus according to morphological characteristic, cell wall and cellular sugar analysis and 16S rDNA sequencing. The study implies a novel chitinolytic actinomycete which could be developed to be a biological agent which would be included as a complement with organic fertilizers in order to control stem rot disease and promote growth of chilli.

Antagonistic Actinomycetes Mediated Resistance in Solanum lycopersicon Mill. Against Rhizoctonia solani Kühn

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Full-text available

Oct 2015

Actinomycetes are a major group of beneficial microbes, which can be explored as spanking alternative to chemical fungicides for providing defense against phytopathogens. Rhizoctonia solani is a major havoc causing severe loss to many crops. Biological measures for fungal disease management are desired over the available chemical/synthetic fungicides owing to their safety towards non-target organisms. In the present study, 34 actinomycetes were isolated from vermicompost. Out of them, twelve revealed antifungal activity related to Indole acetic acid (IAA) production, siderophores and plant growth promotion. Under greenhouse and field conditions, these potent strains remarkably enhanced yield attributes and disease diminution as compared to untreated control. A significant disease reduction of 47–63 % against R. solani was observed in tomato plants pretreated with actinomycetes. Furthermore, induction in defense related enzymes such as peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, accumulation of phenolics and flavonoids were also observed in actinomycetes treated plants. Morphological and molecular characterization analysis identified these potent isolates as Streptomyces sp. NBM3, Streptomyces sp. NBM2, Streptomyces sp. NBM1, Streptomyces sp. NBM12 and Streptomyces sp. NBM8. The present findings suggest that these microbes can be utilized for significant enhancement of plant growth and augmentation of defense related enzymes in order to cope up with R. solani induced stress, thereby contributing to crop health.

Endophytic actinobacteria of medicinal plants: Diversity and bioactivity

Article

Full-text available

Jun 2015
ANTON LEEUW INT J G

Endophytes are the microorganisms that exist inside the plant tissues without having any negative impact on the host plant. Medicinal plants constitute the huge diversity of endophytic actinobacteria of economical importance. These microbes have huge potential to synthesis of numerous novel compounds that can be exploited in pharmaceutical, agricultural and other industries. It is of prime importance to focus the present research on practical utilization of this microbial group in order to find out the solutions to the problems related to health, environment and agriculture. An extensive characterization of diverse population of endophytic actinobacteria associated with medicinal plants can provide a greater insight into the plant-endophyte interactions and evolution of mutualism. In the present review, we have discussed the diversity of endophytic actinobacteria of from medicinal plants their multiple bioactivities.

Rock phosphate-solubilizing Actinomycetes: screening for plant growth-promoting activities

Article

Nov 2008

On the estimation of bound hydroxylamine in biological materials

Article

T.Z. Csaky

Effect of certain fungicides on Sclerotium rolfsii in soil

Article

Jan 1957
PHYTOPATHOLOGY

Ecology of streptomycetes

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Jan 1987

Sclerotium (Athelia) Rolfsii, a Pathogen of Many Plant Species

Chapter

Dec 1988

Zamir Punja

Evaluation of antagonistic and plant growth promoting activities of chitinolytic endophytic actinomycetes associated with medicinal plants against Sclerotium rolfsii in chickpea

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