WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Nithya et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 7.632

Volume 9, Issue 4, 1385-1394 Research Article ISSN 2278 – 4357

ISOLATION AND CHARACTERIZATION OF STREPTOMYCES SPP.

FROM TURMERIC PLANTATION SOILS FOR ANTAGONISTIC
ACTIVITY AGAINST PYTHIUM APHANIDERMATUM, A CAUSAL
PATHOGENIC ORGANISM OF RHIZOME ROT DISEASE OF
TURMERIC PLANTS

M. Nithya*1, P. Ponmurugan2 and B. Mythili Gnanamangai3

1
Research and Development Centre, Bharathiar University, Coimbatore - 641 046, Tamil
Nadu, India.
2
Department of Botany, Bharathiar University, Coimbatore - 641 046, Tamil Nadu, India.
3
Department of Biotechnology, K.S.R. College of Technology, Tiruchengode - 637 215,
Tamil Nadu, India.

Article Received on
ABSTRACT
16 Feb. 2020, An attempt was made to isolate and characterize subsequently identify
Revised on 08 March 2020,
Accepted on 29 March 2020 Streptomyces spp. from turmeric plantation soils collected from
DOI: 10.20959/wjpps20204-15962 different agroclimatic zones of Tamil Nadu, India for the present study
with respect to the production of bioactive secondary metabolites. Soil

*Corresponding Author samples were collected from different places such as Coimbatore,
M. Nithya Dharmapuri, Erode, Salem and Viluppuram districts of Tamil Nadu in
Research and Development which 100 soil samples were collected and subjected to screen for the
Centre, Bharathiar
isolation of Streptomyces spp. The results indicated that the population
University, Coimbatore - 641
density of Streptomyces spp. in turmeric soils was found to be more in
046, Tamil Nadu, India.
Coimbatore (14.7x103 cfu /gm soil dry wt) region followed by Erode
(13.7) and lesser in Viluppuram (9.0) region. The population density of Streptomyces spp.
was interrelated with the nutrient status of turmeric soils which is positive correlated. A total
of 25 isolates of Streptomyces were obtained from soil samples on starch-casein agar and
subjected to purify them for their antagonistic activity against rhizome rot disease causing
pathogenic microorganism Pythium aphanidermatum. Based on the morphological,
biochemical and physiological parameters, a total of five potential isolates were picked up
and identified as Streptomyces spp. Based on the in vitro performance, CS12 isolate was

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promising in terms of antibiotics production when compared to other isolates of

Streptomyces. The results on antagonistic studies indicated that the pathogen’s growth was
suppressed significantly in the in vitro studies and it was found to be of potential antagonist
against P.aphanidermatum.

KEYWORDS: Turmeric soils, Actinomycetes, Streptomyces, Antagonistic activity, Pythium

aphanidermatum.

INTRODUCTION
Turmeric is the most popular spice crop in the world which is produced from rhizomes of the
commercially cultivated turmeric plant (Curcuma longa L) belongs to the family
Zingiberaceae. It is an important profitable plant in India. Indian turmeric is considered the
best in the world in terms of high quality and disease free (Chattopadhyay et al., 2004).
Moreover, it has been reported that turmeric has antimicrobial and anticancerous activities;
besides, biopesticidal and biofungicidal properties are well documented in the current
context. The plant prefers a warm humid climate with well distributed rain fall and moderate
sunshine hours along with temperature and relative humidity. Being an inter-crop,
sometimes; monoculture crop and annual crop pattern of life cycle, it provides a stable
microclimate for harbouring a number of microorganisms in the rhizosphere of turmeric.

Turmeric soil contains a variety of microorganisms and most of them are beneficial to the
plant growth by producing a wide range of growth regulators and antibiotic like substances
(Ponmurugan et al., 2002). Among the soil microorganisms, Streptomyces plays an important
role in terms of production of an array of bioactive secondary metabolites, many of which
have antibacterial or antifungal properties including antagonistic activity (Wellington et al.,
1994) and other inhibitory activities (anti-tumor, anti-fungal, anti-viral) or may function as
herbicides/weedicides (Sanglier et al., 1993). Streptomyces spp. are widely used in industry
due to their ability to produce numerous chemical compounds including antibiotics, enzymes,
organic acids, and anti-tumor compounds as well (Berdy, 1995). The most promising role for
bioactive secondary metabolites extracted from Streptomyces spp. relies upon defense
mechanisms against pathogenic organisms.

Rhizome rot disease caused by a fungal pathogen, Pythium aphanidermatum is a serious

problem in turmeric plantations in different districts of Tamil Nadu, India due to change in
climatic conditions and lack of resistant varieties. There was a significant reduction in

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turmeric export to various foreign countries and also turmeric quality due to this disease
infection (Reddy, 2007, Ponmurugan et al., 2014). Infected plants show a stunted growth,
new leaf turn to yellow in colour with curling of leaf buds. Due to this disease incidence, a
heavy crop loss is recorded which in turn affect the overall quality of the turmeric powder. In
order to control the disease, fungicides like mancozeb and companion are being
recommended by soil drenching method in the soil (Karvy, 2015). Soil drenching of
fungicides lead to deleterious effect on beneficial microorganisms and also affect the soil
health. Further, chemical control is erratic and expensive one. Biological control of rhizome
rot diseases along with plant growth promotion is a viable alternative method of disease
control. In this contest, Streptomyces spp. belongs to actinomycete group is the best candidate
for controlling the plant pathogen (Chang et al., 2006; Fernando et al., 2007). The present
study aims at the isolation, screening and characterization of biologically diverse strains of
Streptomyces from turmeric soils for the production of bioactive secondary metabolites
subsequently tested against P. aphanidermatum.

MATERIALS AND METHODS

Collection of soil samples
Soil samples were collected from different turmeric planting districts of Tamil Nadu such as
Coimbatore, Dharmapuri, Erode, Salem and Viluppuram for isolation of Streptomyces spp.
for the present study. The rhizosphere soil samples were obtained from different turmeric
fields planted with 'Alleppey finger’ variety at a depth of 3-5 cm. These samples were
allowed to air dry at room temperature and various parameters like soil pH, total organic
carbon (Walkley and Black, 1934), total nitrogen (AOAC, 1990) and available phosphorous
(Jackson, 1973) were determined subsequently.

Isolation and characterization of Streptomyces spp.

Enumeration and isolation of Streptomyces spp. present in these soil samples were performed
by serial dilution plate technique using starch-casein nitrate agar. Biochemical
characterizations such as pigment production, starch hydrolysis, casein hydrolysis, catalase
test, nitrate reduction, indole production, gelatin hydrolysis, and hydrogen sulphide
production were carried out to identify the name of actinomycetes. The colony characters
such as margin, elevation and colour were recorded periodically. Gram’s staining was carried
out using crystal violet solution (Ponmurugan and Gangthraparbhu, 2015).

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Effect of biotic and abiotic factors on growth of actinomycetes

Physiological parameters on the influence of abiotic factors such as pH (6.0 to 9.5) and
temperature (25 to 40°C) and nutrient factors such as carbon and nitrogen sources on growth
of Streptomyces were studied. Seven different carbon compounds such as glucose, fructose,
maltose, sucrose, starch and cellulose and four nitrogen compounds such as ammonium
nitrate, sodium nitrate, potassium nitrate and casein hydrolysate were added by replacing
starch and potassium nitrate respectively in the basal medium (starch-casein nitrate agar). The
inoculated plates were inoculated for 10-15 days depending upon the nature of experiment
(Ponmurugan, 2018).

Antifungal metabolite production (Bauer et al., 1996)

Antibiotic production medium (25g starch, 10g glucose, 2g yeast extract, 3g calcium
carbonate, and one ml of trace solution containing ZnSO4, MnCl2, CuSO4, FeSO4, pH 7.5)
was used for extraction of antifungal compounds. Based on the in vitro performance like
growth pattern and culture characteristics described by Goodfellow et al. (1987), the pure
culture of VA isolate was selected for the study. It was inoculated into 25ml of seed medium
in 250 ml conical flask and kept in a rotary shaker at 220 rpm for 25 days. The culture filtrate
was centrifuged at 11,000 rpm to get a clear solution and filter sterilized.

Testing of culture filtrate by antibiosis method (Dennis and Webster, 1971)

The efficacy of Streptomyces culture filtrate was bioassayed in vitro at 10% level against P.
aphanidermatum. The culture filtrate was mixed with molten, cooled PDA medium, so as to
obtain the required concentration, and dispersed uniformly into petriplates. The plates were
inoculated with 5mm mycelial disc of the pathogen P. aphanidermatum. Pathogen inoculated
in unamended medium served as control. The radial growth of the pathogen was measured till
the pathogens in control plates completely covered the plates. Th per cent inhibition of the
growth of P. aphanidermatum was calculated.

Testing of culture filtrate by paper discs method (Evans et al., 1989)

Sterilized filter paper discs impregnated with Streptomyces broth culture was used as an
antifungal metabolite substance contained in it. It was placed onto the PDA plates on
diametrically opposite points after the pathogen colony grew considerably (20-30 mm). The
plates were incubated under room temperature for 3-5 days and were observed for zone of
inhibition, which indicates a positive reaction for antagonistic activity.

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RESULTS AND DISCUSSION

Total number of Streptomyces population present in turmeric soil samples indicated that the
population was found to be 14.7 x103 cfu/gm soil dry weight in Coimbatore and 9.0 x103
cfu/gm soil dry weight in Viluppuram regions. Total population was found to be more in soil
samples collected from Coimbatore region and lesser in Viluppuram soil samples. The
population density was positively correlated to soil nutrients like total organic carbon, total
nitrogen and available phosphorous contents (Table 1). Similar observations were reported in
tea soil samples by Baby et al. (2002) and Bagyalakshmi et al. (2014) who observed a
correlation between beneficial microorganisms and nutrients in the soil. However, most of the
isolates tend to grow in acidic soils which is an important characteristic feature of
Streptomyces species (Stackebrandt et al., 1991). But this report is contrast to the present
observations because turmeric plants are being cultivated in large scale level which is close to
alkaline soil condition.

A total of 25 isolates of Streptomyces (5 isolates from different planting districts) were

obtained from soil samples and subjected to screen for their antagonistic activity. Out of
these, one isolate from each agroclimatic zone was selected based on the culture studies for
further investigation. The isolates were designated as CS12, DS23, ES101, SS1 and VS47
(Table 1). Morphological and biochemical characteristics of the isolates were studied and
results were presented in the Table 2. The results indicated that the purified isolates of
Streptomyces belonged to Streptomyces spp. as they showed good sporulation with compact,
chalk-like dry colonies of different colour variation from pink to white colour. This is the
characteristic features of Streptomyces (Goodfellow, 1987). All the isolates were found to be
Gram’s positive organism and showed a branched mycelium in their cell morphology similar
to fungal characters (Holt, 1989).

The results on biochemical characterization indicated that pigment production was very well
observed in most of the Streptomyces spp. On the other hand, most of the isolates were
efficient in hydrolyzing starch, gelatin and casein. The results coincided with the report of
Ravel et al. (2000). All the strains were found to be efficient in terms of hydrolyzing starch,
gelatin and casein contents. The strains were showed negative response upon indole
production but positive response on catalase test. Production of hydrogen sulphide and nitrite
reduction showed positive result in majority of the isolates (Table 2).

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The growth of Streptomyces isolates on medium adjusted with different pH revealed that a
better growth was recorded between pH 8.0 and 8.5 (Table 3). This pH level may be
correlated with soil pH which may be used for the growth of organisms. The optimum
temperature for the growth of Streptomyces isolates was 25°C followed by 28°C (Table 3).
Among the different carbon sources tested, starch was found to be suitable for maximum
growth followed by maltose. On the other hand, potassium nitrate and ammonium nitrate
were found to be suitable for optimum growth followed by sodium nitrate and casein
hydrolysate (Table 3). It is due to the complexity of the organisms during the growth.
Production of antifungal metabolites have been known to be influenced by components of
medium and cultural conditions such as pH, temperature, carbon and nitrogen sources
(Augustine et al., 2004).

The results on the antagonistic activity of Streptomyces spp. against P. aphanidermatum

showed that the well-developed inhibition zone was formed around paper discs impregnated
with Streptomyces broth culture. The linear growth of P. aphanidermatum was 38.5 mm on
9th day and it was 17.3 mm on 12th day of incubation (Table 4). The results coincided with the
report of Zahner et al. (1979) and Kanimozhi and Ponmurugan (2013). The formation of
inhibition zone around the pathogenic strain is due to the production of secondary metabolites
by Streptomyces spp. (Demain, 1983; Sanglier et al., 1993) and nutrient depleting
environment (Ponmurugan et al., 2014). The formation of inhibition zone around the
pathogenic strain is purely based on the antifungal properties of Streptomyces spp. The
growth of fungal pathogens such as Aspergillus niger, Fusarium oxysporum, Candida
albicans and Crytococcus humicolus was suppressed significantly using antifungal
metabolites obtained from Streptomyces spp (Augustine et al., 2004). In the present study, P.
aphanidermatum failed to germinate in the medium amended with antibiotic substance
extracted from Streptomyces. Moreover, most of the isolates of Streptomyces spp. were found
to be of potential antagonists against P. aphanidermatum and thus proving the production of
secondary metabolites that has the potential to control variety of pathogens in the soil
ecosystem. Based on these results it can be inferred that Streptomyces spp. can be used as a
soil inoculants to prevent the growth of P. aphanidermatum in turmeric soils which in turn
useful to enhance the turmeric rhizome quality.

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Table 1: Population density of Streptomyces spp. and nutrient status in turmeric

plantation soils collected from different district of Tamil Nadu, India.
Name of Organic Total Available
Designation Population Soil
Agroclimatic carbon nitrogen phosphorous
of strains Density * pH
zones (%) (ppm) (ppm)
Coimbatore CS12 14.7 6.8 5.7 3876 14.7
Dharmapuri DS23 10.0 8.0 4.3 3087 13.5
Erode ES101 13.7 6.8 5.6 3881 14.7
Salem SS1 10.3 8.0 4.3 3037 13.3
Viluppuram VS47 09.0 9.5 3.4 2408 10.3
SE ± 1.23 0.63 0.80 22.73 3.12
CD at P=0.05 5.03 1.32 1.02 26.48 5.00
3
* cfu x 10 cfu/gm soil dry wt.

Table 2: Morphological, physiological and biochemical characterization of Streptomyces

isolates obtained from turmeric plantation soils of Tamil Nadu, India.
Isolates of Streptomyces
S.No. Parameters
CS12 DS23 ES101 SS1 VS47
Spiral spore Cluster of Cluster of
1. Cell morphology Rods Rods
chain spore chain spore chain
Colour of the Grayish Pinkish Pinkish
2. White White
mycelium white white white
3. Gram’s staining ++ ++ ++ + -
4. Pigment production ++ ++ ++ - ++
5. Starch hydrolysis ++ - - ++ ++
6. Casein hydrolysis ++ - - ++ ++
7. Catalase test + ++ + ++ ++
8. Nitrate reduction ++ ++ ++ - -
9. Indole production - - - - -
10. Gelatin hydrolysis ++ + ++ + ++
Hydrogen sulphide
11. ++ ++ ++ - -
production
++ Prominent growth
+ Moderate growth
- No growth

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Table 3: Effect of biotic and nutrient factors on the growth of Streptomyces isolates
obtained from turmeric plantation soils of Tamil Nadu, India.
Isolates of Streptomyces
Parameters
CS12 DS23 ES101 SS1 VS47
Optimum pH 8.3 8.8 8.5 8.5 9.0
Optimum temperature (°C) 25 25 28 27 30
Glucose* ++ + + ++ ++
Fructose* ++ + + ++ ++
Maltose* ++ ++ ++ + +
Sucrose* ++ - - - ++
Starch* ++ ++ ++ ++ ++
Cellulose* ++ ++ - - -
Ammonium nitrate** ++ ++ ++ + ++
Sodium nitrate** ++ + + + ++
Potassium nitrate** ++ ++ ++ ++ ++
Casein hydrolysate** ++ + + + ++
++ Prominent growth
+ Moderate growth
- No growth
* Carbon sources
** Nitrogen sources

Table 4: Antagonistic effect of Streptomyces spp. on Pythium aphanidermatum.

Days after Antibiosis method Paper disc method
inoculation (Radial growth in mm) (Linear growth in mm)
1 0.0 (100) 13.5
2 0.0 (100) 16.0
3 0.0 (100) 21.3
4 0.0 (100) 24.0
5 1.7 (93) 27.5
6 3.5 (88) 30.3
7 5.7 (81) 33.3
8 10.1 (71) 36.7
9 13.3 (65) 38.5
10 14.0 (53) -
11 15.5 (50) -
12 17.3 (48) -
SE ± 0.81 1.22
CD at P=0.05 2.43 2.62
* Since the pathogen is slow growing the experiment was completed within 12 days
Values in parentheses denotes % growth inhibition on radial growth

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