Chitinase and Glucanase Activities of Antagonistic
Streptomyces Spp Isolated From Fired Plots under
Shifting Cultivation In Northeast India
Mukesh K Malviya1, Pankaj Trivedi2 and Anita Pandey3*
1Crop Genetics Improvement and Biotechnology Lab, Guangxi Academy of Agricultural Sciences,
Nanning, 530007, China
2Department of Bioagricultural Sciences and Pest Management, Colarado State University, C034 Plant
Sciences, Fort Collins - CO - 80523-1177
*3Biotechnological Applications, G.B. Pant National Institute of Himalayan Environment and Sustainable
Development, Kosi-Katarmal, Almora, 263 643 Uttarakhand, India
Anita Pandey, Biotechnological Applications, G. B. Pant National Institute of Himalayan Environment and Sustainable Development, Kosi-Katarmal, Almora, 263 643 Uttarakhand, India; E-mail:
Received: 10 November, 2017; Accepted: 19 January, 2018; Published: 31 May 2018
Malviya KM, Trivedi P, Pandey A (2018) Chitinase and Glucanase Activities of Antagonistic Streptomyces Spp Isolated From Fired Plots under Shifting Cultivation In Northeast India. J Adv Res Biotech 3(1):1-7. DOI: http://dx.doi.org/10.15226/2475-4714/3/1/00130
Antagonistic Streptomyces spp (Streptomyces sp NEA55 and
Streptomyces cavourensis NEA5), isolated from fired plots under
shifting cultivation in northeast India, are studied for their chitinase
and glucanase activities. The species showed strong antagonism
against test fungi (Rhizoctonia solani and Cladosporium sp.) in plate
assays. Maximum % inhibition was observed due to the effect of
diffusible compounds produced by these species. Streptomyces
sp. NEA55 showed 54.83 % inhibition against R. solani while
S. cavourensis NEA5 showed up to 50.00 % inhibition against
Cladosporium sp. The inhibitory effect of volatile compounds by
Streptomyces sp NEA55 was recorded up to 50.7 % against R. solani
and 37.50 % against Cladosporium sp. While S. cavourensis NEA5
showed 49.23 % inhibition against R. solani and 34.37 % inhibition
against Cladosporium sp. S. cavourensis NEA5 and Streptomyces sp.
NEA55 produced 0.138 ± 0.006 μg/ml and 0.15 ± 0.004 μg/ml chitinase,
0.22±0.001 μg/ml and 0.25±0.002 μg/ml β 1,3 glucanase, respectively.
Both the species showed maximum chitinase activity at pH 6 and
temperature 50 ºC, while minimum enzyme activity was observed
at pH 10 and temperature 20 ºC. Both the species showed glucanase
activity maximum at pH 7 and temperature 40 ºC and minimum
activity at pH 10 and temperature 20 ºC. Both the species hydrolyzed
glycol–chitin as a substrate in denaturing conditions showing variable
amount of different isoforms. This study demonstrates that the
antagonistic species of Streptomyces survive the fire operations under
Keywords: Streptomyces; Antagonism; Chitinase; Glucanase;
Actinobacteria are most widely distributed and distinct
group of microorganisms in nature. Actinobacteria, Streptomyces
species in particular, has been a broadly exploited group of
microorganisms for the production of important secondary
metabolites and enzymes in the field of medicine and agriculture
[1, 2]. Streptomyces are well known as antifungal biocontrol
agents that inhibit several plant pathogenic fungi . The
antagonistic activity of Streptomyces to fungal pathogens is
usually related to the production of antifungal compounds
and extracellular hydrolytic enzymes [4, 5]. Many species of
Streptomyces are well known as antifungal biocontrol agents that
inhibit several plant pathogenic fungi e.g., Phytophthora capsici
Sclerotinia rolfsii, Fusarium sporotrichiodes, Rhizoctonia solani
and Sclerotium rolfsii, Alternaria alternata and Phomopsis archeri
[6-9]. Furthermore, Streptomyces produce bioactive compounds
such as antimicrobial, antiparasitic and immune-suppressing
compounds via secondary metabolism. Streptomycetes have been
found in beneficial associations with plants where they improve
plant growth and protect against pests; this has attracted the
attention of researchers worldwide .
Chitinase and β-1,3-glucanase are considered to be
important hydrolytic enzymes in the lysis of fungal cell walls
. Chitinolytic enzymes have been identified in several
Streptomyces spp. including Streptomyces sp. M-20, S. venezuelae
P10, and S. anulatus CS242 [12-14]. Glucanase has been known
to be produced by several microorganisms and playing important
role in biocontrol . Several Streptomyces have been studied
for antifungal properties along with the production of glucanase,
some of the examples are Streptomyces sp. S27 and Streptomyces
sp. Mo [16, 17]. Shifting cultivation, refers to ‘slash and burn’, is
a predominant form of agricultural practice in hills of northeast
India. The microbiological aspects, basically survival of bacterial,
fungal and actinobacterial communities after fire events have been
studied in recent times [18-20]. The focus of present study is on
the antagonistic potential of two Streptomyces species that were
isolated after the fire events. These species have been studied
with respect to production of diffusible and volatile compounds
against test fungi along with the chitinase and β-1, 3-glucanase
activities. In addition, both the species are also studied with
hydrolyzed glycol–chitin as a substrate in denaturing conditions.
Material and Methods
Study site and isolation of actinobacteria
Actinobacteria were isolated from the soil samples that
were collected after the completion of fire events in Papumpare
District, Itanagar, Arunachal Pradesh under shifting cultivation.
The details of the study sites have been described in Pandey
et al. . Among the two actinobacterial isolates used in the
present study, isolate no. NEA5 showed maximum similarity
with Streptomyces cavourensis NR_043851 and NEA55 with
Streptomyces sp. YIM8 AF389344 . The test fungi (Rhizoctonia
solani and Cladosporium sp.) were also originally isolated from
shifting cultivation site .
Scanning electron microscopy
In order to see the deep morphological pattern (substrate and
aerial mycelia) of Streptomyces spp. scanning electron microscopy
was performed. Glutaraldehyde (2.5 %) was added to the culture
and then centrifuged. After centrifugation, washing was given
with phosphate buffer saline (PBS) twice and centrifuged. The
sample was dehydrated with CPD (Critical dry point), and then
the samples were coated with gold by auto fine coater (JFC-1600).
After coating, the sample was viewed under scanning electron
Dual culture technique for determination of the production of
diffusible antifungal compounds
To test the ability of the Streptomyces spp. to inhibit the
phytopathogens, the test fungal culture and Streptomyces sp. was
spot inoculated off-center on each potato carrot agar (PCA) plate.
After 7 days of incubation at 28 ºC, the zone of inhibition was
measured. Per cent growth inhibition was calculated using the
following formula: (R1-R2)/R1 × 100 (where R1 represents the
radius of the test fungus in the direction with no bacterial colony
and R2 is the radius of the fungal colony in the direction of the
bacterial colony) as described in Trivedi et al. .
Sealed double Petri plate technique for determination of the
production of volatile antifungal compounds
Each Streptomyces sp. was streaked on Petri plate containing
PCA and a 6.0 mm disc of 4 days old culture of the test fungus
was placed in the middle of another PCA plate. The lid of both the
plates were replaced with the plates of same diameter, placed face
to face, and sealed by parafilm, preventing any physical contact
between the pathogen and the Streptomyces sp. Petri plates were
incubated at 28 ºC for 7 days and growth of the pathogen was
measured and compared to control developed in the absence
of the antagonist (mocked inoculation with a 6.0 mm disc of
PCA). Results were expressed as means of per cent inhibition
of the growth of each fungus in the presence or absence of the
antagonistic Streptomyces sp. Per cent growth inhibition was
calculated by the following formula: (R1-R2)/R1 × 100, where R1
represents the diameter of the test fungus on control plate and R2
is the diameter of the growth on inoculated plate.
Quantitative estimation of production of chitinase and
glucanase on different temperature and pH
Quantification of chitinase and glucanase has been done
following prescribed procedures . The Streptomyces species
were cultured at 28 ºC for 5 days on a rotary shaker in 250.0
ml of chitin peptone medium for chitinase production and
peptone medium containing laminarin (0.2 %) (from Laminaria
digitata, Sigma) for glucanase production . The cultures were
centrifuged at 12,000 g for 20 min at 4 ºC and the supernatants
were used as enzyme source. The reaction mixture contained 0.25
ml of enzyme solution, 0.3 ml of pH buffer (1.0 M citrate buffer
pH 5, phosphate buffer pH 6 to 8, and glycine buffer pH 9). The
reaction mixtures were incubated at different temperatures (20,
30, 40, 50 and 60 ºC) for 4 h in a water bath. One unit of chitinase
was determined as 1 μmol of N acetyl glucosamine (GlcNAc)
released min-1 and β-1, 3-glucanase activity was determined as 1
μmol of glucose released min-1. Protein content was determined
as described by Lowry method .
SDS-PAGE analysis for chitinase
Streptomyces spp. was grown as shake culture in chitinpeptone
medium at 28 ºC for 7 days. The supernatant was
centrifuged at 12000 g for 20 min at 4 ºC and filtered through 0.22
μl sterile filter (Millipore) and collected in conical flask. Protein
content was analyzed by SDS-PAGE . Polyacrylamide slab gel
consisted of 4 % stacking gel and 10 % separating gel containing
0.01 % glycol–chitin. Electrophoresis was carried out at a
constant voltage of 65 V; gel was stained with coomassie brilliant
blue R-250 and analyzed under gel documentation system (Alpha
Imager 2200). Glycol chitin was prepared as described by Trudel
and Asselin .
Results and Discussion
The scanning electron microscopy of both the species is
presented in Figure: 1A and Figure: 1B. Scanning electron
microscopy revealed definite structures of mycelium and spores
of both the Streptomyces spp. Two of the isolates that were used
for detailed studies based on their antagonistic properties were
subjected to scanning electron microscopy. The scanning electron
microscopy has been referred to provide perfect characterization
of streptomycetes . The effect of diffusible compounds
produced by the Streptomyces spp., evaluated in terms of
reduction in radial growth of two test fungi viz. Rhizoctonia solani
and Cladosporium sp., following 7 days of incubation at 28 ºC, are
presented in Figure:2 & Table 1. The results showed that volatile
compounds produced by Streptomyces spp. were inhibitory
to the growth of the test fungi, viz. R. solani and Cladosporium
sp. Inhibition (%) in fungal growth by Streptomyces species
is presented in Table 1. Microscopic observations revealed
that the diffusible as well as volatile compounds produced by
Figure 1A: Scanning electron micrograph of Streptomyces cavourensis NEA5
Figure 1B: Scanning electron micrograph of Streptomyces sp. NEA55
Figure 2: Production of antifungal compounds by Streptomyces cavourensis NEA5 & Streptomyces sp. NEA55 (A&B) respectively: Inhibition of R. solani and Cladosporium sp. due to diffusible compounds produced. Morphological deformities: (A1&B1) Normal structures of R. soloni and Cladosporium sp.(A2 & B2) deformed structures of respective fungus. (Bar = 5 μm)
Table 1: In vitro percent inhibitory effect of diffusible and volatile metabolites of Streptomyces species on growth of test fungi
Pathogenic test fungi
% Inhibition in fungal growth by Streptomyces spp. after 7 days of incubation
Streptomyces cavourensis NEA5
Streptomyces sp NEA55
Table 2: Comparative account of structures of normal and antagonized pathogen
Somatic and fertile hypha well distinguished
Septate, dia. 2.5- 3.5 µm
septate, dia 3.0-6.0
Septate and dark in colour, dia. 3.0-4.5 µm
Irregular septate, dia 3.0-7.5 µm
Somatic hypha septate, dia. 2-3.5 µm
Somatic hypha septate, dia. 2-5 µm
Curved or straight width 2.0-2.5 µm
Irregular width 2-4 µm
Streptomyces species induced morphological abnormalities in the
fungal structures (Table 2). Deformation was observed in mycelial,
hyphal or conidial structures. The longitudinal septae completely
disappeared and the conidia became thick walled and spherical
or irregular in shape. Lysis of fungal hyhae and vacuolization as
well as granulation in mycelium was observed in R. solani. Size
of the somatic and fertile hypha of R. solani increased due to the
antagonistic effect of actinobacteria. Due to the antifungal activity
of Streptomyces sp., hyphal wall of R. solani was completely
lysed. In case of Cladosporium sp., size of mycelium and conidia
increased with irregular shape of conidia. The inhibition of
growth of the test pathogenic fungi continued to increase with
increasing incubation time. A clear inhibitory area and dense
sporulation ring was observed near the growth of the isolate
which antagonized respective test fungi. No physical contact was
observed between the Streptomyces species and the test fungi that
were observed to be antagonized. Moreover, the formation of the
inhibitory halo suggested the presence of fungicidal metabolites
secreted by Streptomyces. Similar observations have been
reported by Aghigni et al.  from a number of actinobacterial
isolates from Iranian soil. These isolates formed inhibition zones
in dual culture based assays inhibiting the growth of Alternaria
solani, A. alternata, Fusarium solani, Phytophthora megaperma,
Verticillium dahlia and Saccharomyces cerevisiae. The antifungal
potential of actinobacteria against Colletotrichum gloeosporioides
and Sclerotium rolfsii was assessed by dual culture technique .
Al-Askar et al.  reported that Streptomyces spororaveus RDS28
produces antifungal compounds against some phytopathogenic
fungi, viz., Rhizoctonia solani, Fusarium solani, F. verticillioides,
Alternaria alternata and Botrytis cinerea. There was a change
in colour of the test fungi in inoculated plates, indicative of the
inhibitory effect of volatile compounds. The test fungi, inhibited
by the Streptomyces, under present study exhibited morphological
abnormalities due to the production of diffusible and volatile
antifungal compounds. Abnormal hyphal swelling, degradation
and lysis of mycelia were observed by Joo  when Phtophthora
capsici was grown with the high or low molecular fraction of
Streptomyces halstedii AJ-7.
Quantitative estimation of production of chitinase and β 1,
3 glucanase activities is presented in Figure 3 (A&B) showing
maximum activity of chitinase and glucanase. Effect of different
pH and temperature on chitinase activity of both the species
is presented in Figure 4 (A&B). Both the species showed
maximum activity of chitinase at pH 6 and temperature 50 ºC,
while minimum enzyme activity was observed at pH 10 and
temperature 20 ºC. In case of glucanase activity both the species
showed maximum enzyme activity at pH 7 and temperature 40
ºC and minimum activity at pH 10 and temperature 20 ºC (Figure
5A&B). Kim et al.  reported maximum activity of chitinase
Figure 3A: Quantitative estimation of chitinase activity
Figure 3B: Quantitative estimation of glucanase activity
Figure 4A: Chitinase activity of Streptomyces cavourensis NEA5 at different pH & temperature
Figure 4B: Chitinase activity of Streptomyces sp NEA55 at different pH & temperature
Figure 5A: Glucanase activity of Streptomyces cavourensis NEA5 at different pH & temperature
between pH 5.0 to 6.0 and it was relatively stable at pH 4.0 to 8.0
when kept at 4 ºC. Beyond this pH range there was a rapid loss
in the activity. Glucanase activity of the two isolates was found
maximum at pH 7 and temperature 40 ºC. Highest activity was
found between pH 5 to 8 and temperature 30 to 50 ºC, in previous
studies. The optimum activity for short term incubation is often
seen at temperature in the range of 30 to 50 ºC, while many fungal
glucanases appear stable between 50 to 60 ºC .
SDS-PAGE exhibited the chitinase activity of the two
Streptomyces species (NEA5 & NEA55). Streptomyces species
hydrolyzed glycol-chitin as a substrate in denaturing conditions.
Species showing variable amount of different isoforms with
molecular weights between 31 to 40 kDa are presented in Figure
6. Majority of bacterial chitinases has been reported to be in the
range of 20 to 60 kDa. Chitinases from various Streptomyces were
found to possess molecular weights as 20 kDa from Streptomyces
Figure 5B: Glucanase activity of Streptomyces sp NEA55 at different pH & temperature
Figure 6: Detection of chitinase activity after SDS-PAGE using glycol
chitin as substrate, lane 1 (Molecular weight markers), lane 2 and 3
sample Streptomyces cavourensis NEA5 and Streptomyces sp NEA55, respectively
sp. M-20  and some strains of Streptomyces varying from
approximately 25 kDa to 200 kDa .
The aim of this work is to prove the potential of Streptomyces
species to survive the fire operations retaining their potential
to reduce or eliminate the plant pathogenic fungi. Such robust
isolates may prove to be the potential biocontrol agents for field
applications. Further studies are needed in order to determinate
the nature of Streptomyces species metabolites and their
mechanism of action.
Director (GBPNIHESD) for extending the facilities and
Ministry of Environment, Forest and Climate Change, Govt. of
India, New Delhi for financial support.
- Kumar D, Gupta RK. Biocontrol of wood rotting fungi. Ind J Biotechnol. 2006; 5:20-25.
- Narayana KJP, Vijayalakshmi M. Chitinase production by streptomyces sp. anu 6277. Braz J Microbiol. 2009; 40(4): 725-733.
- El-Tarabily KA, Soliman MH, Nassar AH, Al-Hassani HA, Sivasithamparam K. Biocontrol of sclerotinia minor using a chitinolytic bacterium and actinomycetes. Plant Pathol. 2000; 49: 573-583.
- Fourati-Ben Fguira L, Fotso S, Ben Ameur-Mehdi R, Mellouli L, Laatsch H. Purification and structure elucidation of antifungal and antibacterial activities of newly isolated Streptomyces sp. Strain US80. Res Microbiol. 2005; 156(3): 341-347.
- Trejo-Estrada SR, Sepulveda IR, Crawford DL. In vitro and in vivo antagonism of Streptomyces violaceusniger YCED9 against fungal pathogens of turfgrass. World J Microbiol Biotechnol. 1998;14(6):865-872.
- Joo GJ. Production of an anti-fungal substance for biological control of Phytophthora capsici causing phytophthora blight in red-peppers by Streptomyces halstedii. Biotechnol Lett. 2005;27(3):201-205.
- Errakhi R, Bouteau F, Lebrihi A, Barakate M. Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.). World J Microbiol Biotechnol. 2007;23(11):1503-1509.
- Rugthaworn P, Uraiwan Dilokkunanant , Sangchote S, Piadang N, Kitpreechavanich V. Search and improvement of actinomycete strains for biological control of plant pathogens. Kasetsart J (Nat Sci). 2007;41:248-254.
- Malviya MK, Pandey A, Trivedi P, Gupta G, Kumar B. Chitinolytic activity of cold tolerant antagonistic species of streptomyces isolated from glacial sites of Indian Himalaya. Curr Microbiol. 2009;59(5):502-508. doi: 10.1007/s00284-009-9466-z
- Sousa JAJ, Olivares FL. Plant growth promotion by streptomycetes: ecophysiology, mechanisms and applications. Chem Biol Technol Agric. 2016; 3:24.
- Prapagdee B, Kuekulvong C, Mongkolsuk S. Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi. Int J Biol Sci. 2008;4(5):330-337.
- Kim KJ, Yang YJ, Kim JG. Purification and characterization of chitinase from Streptomyces sp. M-20. J Biochem Mole Biol. 2003;36(2):185-189.
- Mukherjee G, Sen SK. Purification, characterization, and antifungal activity of chitinase from Streptomyces venezuelae P10. Curr Microbiol. 2006;53(4):265-269.
- Mander P, Cho SS, Choi YH, Panthi S, Choi YS, Kim HM, Yoo JC et al. Purification and characterization of chitinase showing antifungal and biodegradation properties obtained from Streptomyces anulatus CS242. Arch Pharm Res. 2016;39(7):878-886. doi: 10.1007/s12272-016-0747-3
- El-Katatny MH, Gudelj M, Robra KH, Elnaghy MA, Gubitz GM. Characterization of a chitinase and an endo- β-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii. Appl Microbiol Biotechnol. 2001; 56(1-2):137-143.
- Shi P, Yao G, Li N, Luo H, Bai Y, Wang Y, Yao Bin et al. Cloning, characterization, and antifungal activity of an endo-1,3-β-D: -glucanase from Streptomyces sp. S27. Appl Microbiol Biotechnol. 2010;85(5):1483-1490. doi: 10.1007/s00253-009-2187-1
- Kurakake M, Yamanouchi Y, Kinohara K, Moriyama S. Enzymatic properties of β-1,3-glucanase from Streptomyces sp Mo. J Food Sci. 2013;78(4):502-506. doi: 10.1111/1750-3841.12076
- Pandey A, Chaudhry S, Sharma A, Choudhary VS, Malviya MK, Chamoli S, Rinu K, Trivedi P, Palni LM et al. Recovery of Bacillus and Pseudomonas spp from the ‘Fired Plots’ under Shifting Cultivation in Northeast India. Curr Microbiol. 2011; 62(1): 273-280. doi: 10.1007/s00284-010-9702-6
- Malviya MK, Pandey A, Sharma A, Tiwari SC. Characterization and identification of actinomycetes isolated from ‘fired plots’ under shifting cultivation in northeast Himalaya, India. Ann Microbiol. 2013;63(2):561-569.
- Jain J, Chaudhary D, Dhakar K, Pandey A. A consortium of fungal survivors after fire operations under shifting cultivation in Northeast Himalaya, India. Nat Acad Sci Lett. 2016;39(5):343-346.
- Trivedi P, Pandey A, Palni LM. In vitro evaluation of antagonistic properties of Pseudomonas corrugata. Microbiol Res. 2008;163(3):329-336.
- Nagarajkumar M, Bhaskaran R, Velazhahan R. Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen. Microbiol Res. 2006;159(1):73-81.
- Lim H, Kim Y, Kim S. Pseudomonas stutzeri YLP-1 genetic transformation and antifungal mechanism against Fusarium solani, an agent of plant root rot. Appl Environ Microbiol. 1991;57(2):510-516.
- Bollag DM, Rozycki MD, Edelstein SJ. Protein methods, 2nd Ed. Wiley New York. 1996:432.
- Laemmli UK. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature (London). 1970;277:680-685.
- Trudel J, Asselin A. Detection of chitinase activity after polyacrylamide gel electrophoresis. Ann Biochem. 1989; 178(2):362-366.
- Dietz A, Mathews J. Scanning Electron Microscopy of Selected Members of the Streptomyces hygroscopicus Group. Appl Microbiol. 1969;18(4):694-696.
- Aghigni S, Boniar GHS, Rowashdeh R, Batayneh S, Saadoun L. First report on antifungal spectra of activity of Iranian actinomycetes strains against Alternaria solani, Alternaria alternata, Fusarium solani, Phytophthora megasperma, Verticillium dahlia and Saccharomyces cerevisiae. Asian J Plant Sci. 2004;3(4):463-471. doi: 10.3923/ajps.2004.463.471
- Al-Askar AA, Abdul Khair WM, Rashad YM. In vitro antifungal activity of Streptomyces spororaveus RDS28 against some phytopathogenic fungi. Afr J Agric Res. 2011;6(12):2835-2842.
- Muskhazli M, Ramli SA, Ithni A, Tohfah NF. Purification and characterisation of β-l, 3-giucanase from Trichodenna harzianum BIO 10671. Pertanika J Trop Agric Sci. 2005; 28(1): 23-31.
- Gomes RC, Semeˆdo LTAS, Soares RMA, Alviano CS, Linhares LF, Coelho RR et al. Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol. Lett Appl Microbiol. 2000; 30(2):146-150.