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Cultural and morphological characterization of Colletotrichum capsici causing

anthracnose of chilli (Capsicum anum L.)

Article · January 2020

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 Journal of Pharmacognosy and Phytochemistry 2020; 9(3): 1985-1989

E-ISSN: 2278-4136
P-ISSN: 2349-8234
www.phytojournal.com Cultural and morphological characterization of
JPP 2020; 9(3): 1985-1989
Received: 10-03-2020 Colletotrichum capsici causing anthracnose of
Accepted: 12-04-2020
chilli (Capsicum anum L.)
Manoj Kumar Prajapati
Department of Plant Pathology,
GBPUAT College of Agriculture, Manoj Kumar Prajapati, Dr. Shilpi Rawat, Priya Singh and Kripa
Pantnagar, Udham Singh Nagar Shankar
Uttarakhand, India

Dr. Shilpi Rawat Abstract

Department of Plant Pathology, Anthracnose of chilli, incited by Colletotrichum capsici, is a major concern for the farmers in various
GBPUAT College of Agriculture, parts of the country. An experiment was undertaken to study the cultural and morphological
Pantnagar, Udham Singh Nagar characterization of the pathogen through in vitro studies. Results revealed that OMA was found to be best
Uttarakhand, India for maximum radial growth (90.00 mm) of the test pathogen followed by CFDA (88.67 mm) whereas
minimum radial growth (69.34 mm) was observed in RSA. Among five different temperature, maximum
Priya Singh radial growth (89.87mm) of the pathogen was recorded at 30 ºC followed by 25 ºC (84.83mm) whereas at
Department of Plant Pathology, 40 ºC, no radial growth was found. Among the five different pH tested, pH 7.0 was found to be best
GBPUAT College of Agriculture,
suited for the radial growth of the pathogen (83.67 mm) followed by pH 8.0 (81.00 mm) whereas
Pantnagar, Udham Singh Nagar
Uttarakhand, India
minimum radial growth was recorded at pH 4.0 (74.33mm). Colony culture of the fungus showed grayish
to white colony colour with fluffy texture and smooth margin in OMA, PDA, CDA, RSA and MEA
Kripa Shankar whereas brown to black with thin scanty mycelium was observed in CFDA. Microscopic observation
Department of Fruit Science, (40X) revealed that mycelium was dense, filamentous and septate, acervuli with brown coloured and
College of Horticulture and rounded. Setae were brown to black in colour long needle like, swollen at base and tapering towards apex
Forestry, Central Agricultural having size of 110-272μm ×4-6 μm with 2-5 septa per setae. Conidia were hyaline, single celled, sickle
University, Pasighat, Arunachal shaped, presence of oil globules with size 18-27 µm × 2.1-4.1 µm.
Pradesh, India
Keywords: Chilli, Colletotrichum capsici, characterization, media, temperature, pH

1. Introduction
Chilli (Capsicum annum L.) is renowned and illustrious all over the world for its spicy taste. It
is an important annual spice as well as vegetable crop belonging to Solanaceae family. The
origin of chilli is considered as Southern American tropics and is currently being cultivated
throughout the world including the tropical, subtropical and temperate regions (Pickersgill,
1997) [15]. Chilli, an important monetary crop worldwide (Poulos, 1992) [16] and sustainability
of chilli production is threatened by various types of biotic factors including fungi, bacteria,
viruses and other pests involving root-knot nematodes, aphids, thrips and weeds and abiotic
factors involve light, temperature, rainfall, herbicides, pesticides which cause directly or
indirectly significant yield losses in chilli production. The diseases like anthracnose, bacterial
wilt, chilli mosaic, leaf curl and several insect pests have been reported to reduce the crop
productivity (Issac, 1992; Anand et al., 2010) [8, 5]. But it is severely exaggerated by
anthracnose disease which is one of the major economic constraints to chilli production
worldwide, especially in tropical and sub-tropical regions (Than et al., 2008) [20] which may
cause yield losses up to 50 per cent (Pakdeevaraporn et al., 2005) [14] and 10–80% loss due to
pre and post harvest disease (Than et al., 2008) [20]. Colletotrichum capsici produces colony
colour with white to grey having dark green centre. Initially mycelium is hyaline, richly-
branched, dense, filamentous, septate later on become dark at maturity (Than et al., 2008) [20].
The acervuli are saucer-shaped and surrounded by firm, black, unbranched hairs like structure
typically referred to as setae. Conidia are colourless, one- celled and shape is varying from
sickle shaped, ovoid, cylindrical size of conidia is around to 17-18 x 3-4 µm. (Agrios, 2005)
[2]
. Anthracnose is characterized by circular or angular, depressed, sunken lesions and
concentric rings of acervuli and producing pink to orange conidial masses (Isaac, 1992 and Oo
et al., 2016) [8, 13]. Generally, warm, wet climate (rainy weather) along with temperature
Corresponding Author: approx 27 °C, RH 75 to 80% and soil pH 5 to 6 favours the disease development. (Roberts et
Manoj Kumar Prajapati
Department of Plant Pathology,
al., 2001 and Rashid et al., 2015) [19, 8]. Keeping in view the importance of the crop losses
GBPUAT College of Agriculture, caused by this devastating pathogen the present investigation has been carried out on cultural
Pantnagar, Udham Singh Nagar and morphological characterization of the Colletotrichum capsici causing anthracnose of chilli.
Uttarakhand, India
~ 1985 ~
Journal of Pharmacognosy and Phytochemistry http://www.phytojournal.com

2. Materials and Methods constant. The test fungus was grown on autoclaved, molten
2.1 Collection, isolation and identification and cooled medium were poured aseptically on to sterilized
2.1.1. Isolation of test pathogen petri plates and allowed for solidification of the media, and
Experiments were conducted during the crop season 2018- then each plates were centrally inoculated with 5 mm culture
2019 for cultural and morphological characterization. disc of pathogen cut from the margins of 7 to 10 days old
Laboratory experiments were carried out in Department of culture by using sterilized cork borer under aseptic condition
Plant Pathology G.B. Pant University of Agriculture and then incubated at 28±2 oC in BOD incubator. Pathogen was
Technology, Pantnagar, Udham Singh Nagar (Uttarakhand). identified for its morphological structures such as acervuli,
The pathogen was isolated from the freshly infected fruits presence and absence of setae septation and conidia shape and
through standard tissue isolation technique.The pure culture size and presence of oil globules were examined under stereo-
of the pathogen was obtained by single hyphal tip isolation binocular microscope as well as electron microscope.
technique. Pure cultures were maintained on PDA slants at
4oC in refrigerator and sub cultured on petri plates containing 3. Statistical Analysis
potato dextrose agar medium for further experiments. The data analysis was performed by STPR software.

2.1.2. Effect of different media, temperature and pH on 4. Results and Discussion

radial growth and colony characterization of test 4.1 Colony characters of the test pathogen on different
pathogen media
The in vitro studies were laid out in completely randomized The data presented in table 1 revealed that, in Potato Dextrose
design with three replication each. Observation was recorded Agar media, fungus showed grayish to white colony colour
daily for 9 days. Six different media, three synthetic, i.e. with thin, scanty texture and smooth margin whereas in Chilli
Richardʼs synthetic agar media, Malt extract and Czapekʼs- Fruit Decoction Agar media, colony colour was dark brown to
dox, and three semisynthetic, i.e. Potato Dextrose Agar, Oat black, circular with thin scanty texture and smooth margin. In
Meal Agar Media and Chilli Fruit Decoction Agar Media, Oat Meal Agar media, dull white to grey colony colour was
were used to study the radial growth of the test pathogen as observed having compact texture with smooth margin and
well as colony characteristics of C. capsici such as colour, prominent zonation. In Czapedox Agar media, buff whitish
texture and margin. The characteristics were compared with colony colour along with fluffy texture and smooth margin
the colour chart (Akhtar and Singh, 2007). Similarly, the was observed. Dull white to gray colony colour, depression at
effect of temperature on the radial growth of the pathogen was the centre with slightly fluffy growth at the outer side and
studied at different temperature ranges viz. 20, 25, 30, 35 and smooth margin was observed in Richards Synthetic Agar
40 oC and pH levels viz., 4.0, 5.0, 6.0, 7.0 and 8.0 which were Media. White to greyish colony colour with slightly fluffy
adjusted with the help of digital pH meter using 0.1N NaOH texture and serrete type of margin was observed in Malt
or 0.1N HCl before autoclaving for maintaining the pH Extract Agar media (Plate 1).

Table 1: Colony characteristics of the C. capsici on different media

Colony character
S. No Media
Color Texture Margin
1 Potato Dextrose Agar Grayish white Thin scanty Smooth
2 Oat Meal Agar Dull white to grey Compact Smooth
3 Chilli Fruit Decoction Agar Dark brown to black Thin scanty Smooth
4 Czapekʼs dox Agar Buff whitish Fluffy Smooth
5 Richardʼs Synthetic Agar Dull white to grey Slightly fluffy Smooth
6 Malt Extract Agar White to greyish Slightly fluffy Serrete

4.2 Morphological characteristics of the test pathogen 345.17 µm in length and width of 2.82 -5.74 µm with 2-5
Morphological characteristics under stereo-binocular septa per setae. Conidia are sickle shaped with narrower end
microscope (40X) revealed that mycelium was dense, and broad centre, size varying between 6.17 -6.7 µm ×1.6-
filamentous and septate, acervuli were dark brown, rounded, 1.78 µm at 2000X with a depression at the centre (Plate 2).
elongated. Setae were dark brown to black in colour long These research findings are in accordance with Kulshrestha et
needle like structure swollen at base and narrow at the end al. (1976) [11] and Akhtar and Singh (2007) [3] they reported
with 110-272 μm in length and 4-6 μm in width with 2-5 that the mycelium of Colletotrichum capsici was fine, shiny
septa per setae. Profuse thick walled conidia which were or whitish pink in colour. They described Colletotrichum
hyaline, uninucleate, falcate shape (sickle shaped) slightly capsici based on relative size of the setae and shape of conidia
tapering or rounded towards end with presence of oil globules in relation to the conidial mass in acervuli. Acervuli were
in centre and measured 18-27 µm × 1.8-4.1 µm in size (mean single or in groups. Setae numerous, blackish brown to dark
21.64×2.85 µm). As observed under electron microscopic black, longer than the conidial mass. Conidial mass was white
pictograph acervuli measured approximately 39.84 µm in to dull white, pale orange or bright orange. Conidia hyaline,
diameter at 550X. Long needle like structure of setae emerge fusoid, ends rounded or slightly tapering and having 15-27 x
out from the ruptured acervuli. Setaes were swollen at the 2-5 μm in size. Setae have 0-9 septa and 48-468 x 2-7 μm in
base and tapering towards apex having size of 107.74 µm to size.

~ 1986 ~
Journal of Pharmacognosy and Phytochemistry http://www.phytojournal.com

a). Inoculated fruit b). Acervuli on fruit c). Presence of setae d). Culture plate

e). Hyphae f). Mycelium g). Profuse conidia h). Septa

i). Enlarge view of setae at 100X j). Conidia with oil globule k). Measurement of setae l). Single setae
Fig 1: Morphological characteristics of the C. capsici

4.3 Effect of different media on radial growth of C. capsici capsici and full plate growth (90.00 mm) observed within 9
The data presented in table 2 revealed that maximum radial days followed by YEPD, CEA, WEA and MEA. The reason
growth (90.00 mm) was observed on 9 days in OMA followed may be difference in the isolates of C. capsici and its
by CFDA (88.67 mm) which were statistically at par to each selectivity for the media. Growth of mycelium and
other and differ significantly with rest of media tested. Other sporulation are affected by different types of media. Media
mediaʼs viz. PDA showed radial growth of 82.67 mm contains carbohydrate, lipid, protein and elements are basic
followed by CDA with 80.16 mm, RSA with 69.34 mm where requirements and needed by the microorganisms to provide
in PDA and CDA were found to be statistically at par with energy for biosynthesis and cell maintenance and production
each other. Minimum radial growth of 53.33 mm was of biomass in fungi and growth-associated products requires
observed in RSA. (Figure2).These results are in accordance nutrient-balanced media (Hilton, 1999) [7]. In OMA, presence
with the findings of Javed (2014) [9]. who reported that of high amount of carbohydrate, proteins and lipids as
maximum radial growth of colony of C. capsici was in Oat compared to other media which is responsible for fast radial
Meal Agar and Corn Meal Agar, followed by PDA and growth of the fungus. Vega et al. (2003) [22] reported that
Richard’s agar whereas Admassie et al. (2015) [1] reported some dimorphic fungi require optimal nutrition to produce
that maximum radial growth of C. capsici was observed on high biomass, but for sporulation require nutritionally poor
pepper dextrose agar (prepared from leaves, stems and fruits media which trigger differentiation of conidia from vegetative
of pepper) and potato dextrose agar medium. However, these growth. In spite of containing parts or chemical substances
research findings are contradictory with research finding of from the fungus, natural host and natural media do not always
Akhtar and Singh (2007) [3] who reported that PDA was found elicit the best sporulation and similar results were also
best suited media for mycelial growth of Colletotrichum observed by Pria et al. (1997) [17] and Hanada et al. (2002) [6].

a) OMA b) CFDA c) PDA d) CZPX e) RSA f) MEA

Fig 2: Colony characters and radial growth of C. capsici on different media.
~ 1987 ~
Journal of Pharmacognosy and Phytochemistry http://www.phytojournal.com

Table 2: Effect of different media on radial growth of C. capsici incubation. The data presented in table 3 revealed that
Colony diameter maximum mycelial growth was recorded at 30 ºC (89.87 mm)
T.N. Media followed by 25 ºC with 84.83 mm which differ significantly.
(mm)*
T1 Potato Dextrose Agar Medium (PDA) 82.67 At 35 ºC mycelial growth of 79.67 mm and at 20 ºC mycelial
T2 Oat Meal Agar Medium (OMA) 90.00 growth was observed 75.33 mm whereas at 40 ºC, no radial
T3 Chilli Fruit Decoction Agar (CFDA) 88.67 growth was found after 9 days of incubation. All the
T4 Czapekʼs Dox Agar Medium (CZPX) 80.16 temperatures were statically significantly different with each
T5 Malt Extract Agar Medium (MEA) 53.33 other and temperature range of 25-35 ºC is optimum for the
Richardʼs Synthetic Agar Medium mycelial growth of the fungus (Figure 3). These results are
T6 69.34
(RSA) confirmative with research findings of Admassie et al. (2015)
SEm± = 1.11 [1]
, Tripathi et al. (2016) [21], Kommula et al. (2017) [10] and
CD at 5% = 3.42 Akhtar et al. (2018) [4] they reported that the maximum
CV = 2.48
mycelial growth of C. capsici was found at temperature range
* represents average of three replication
between 25-30 °C. Majority of the fungi require optimum
temperature ranges of 25 to 30 ºC for their mycelial growth,
4.4 Effect of temperature on radial growth of C. capsici
whereas at high temperature (40 ºC) disintegration of cell
The growth rate of the pathogen was recorded at five different
wall, protein and enzymes lysis may occur.
temperatures viz; 20, 25, 30, 35 and 40 ºC for 9 days of

a) 20 OC b) 25 OC c) 30 OC d) 35 OC e) 40 OC
Fig 3: Effect of different temperature on radial growth of C. capsici

Table 3: Effect of different temperature on radial growth of C. statistically significant with each other (Plate 4).The research
capsici finding of Akhtar et al. (2018) [4] who reported that maximum
Tr. No. Temperature (ºC) Colony Diameter (mm)* radial growth (90.00 mm) of pathogen was observed at pH 7.0
T1 20 75.33 followed by 8.0, 6.0, 5.0 and 4.0. Kommula et al. (2017) [10]
T2 25 84.83 studied the effect of different pH levels viz. 3.0, 3.5, 4.0, 4.5,
T3 30 89.87 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 and found that maximum growth
T4 35 79.67 of the pathogen was observed at pH range of 6.5-7.0 and
T5 40 0.00 minimum growth was recorded at pH 3.0. Thus it can be
SEm± = 0.07 concluded that cultural and morphological characterization of
CD at 5% = 0.23 the pathogen provide better understanding about the pathogen
CV = 0.19 biology and etiology through which integrated approach for
* represents the average of three replication disease management can be effort.
4.5 Effect of pH on the radial growth of the C. capsici Table 4: Effect of pH on radial growth of the C. capsici
In vitro evaluation of five different pH levels viz., 4.0, 5.0,
6.0, 7.0 and 8.0 to find out suitable pH for the radial growth Tr. No pH Colony diameter (mm)*
of the pathogen and observation were taken daily for 9 days T1 4.0 74.33
of incubation. The data presented in table 4 revealed that pH T2 5.0 76.50
7.0 was found to be best suited for the growth of the pathogen T3 6.0 78.70
with a significant maximum colony diameter of 83.67 mm T4 7.0 83.67
followed by radial growth of 81.00 mm, 78.70 mm and 76.50 T5 8.0 81.00
mm with pH of 8.0, 6.0 and 5.0 respectively. Least radial SEm = 0.15
growth was recorded at pH 4.0 with 74.33 mm only. Among CD at 5% = 0.48
CV = 0.32
all pH, the radial growth of the pathogen was found to be
*represents the average of three replication

a) pH 4.0 b) pH 5.0 c) pH 6.0 d) pH 7.0 e) pH 8.0

Fig 4: Effect of pH on radial growth of C. capsici
~ 1988 ~
Journal of Pharmacognosy and Phytochemistry http://www.phytojournal.com

5. Summary and Conclusion 13. Oo MM, Oh SK. Chilli anthracnose (Colletotrichum spp.)
The present experiment was conducted to figure out the disease and its management approach. Korean Journal of
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~ 1989 ~

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