Screening and Potential for Biological Control of

Anthracnose Disease (Colletotrichum capsici) on

Chili Fruits by Yeast Isolates

Punika Chaisemsaeng, Wiyada Mongkolthanaruk, and Wandee Bunyatratchata Department of Microbiology/Faculty of Science/Khon Kaen University, 40002, Thailand

Email: cpunika@yahoo.com,{wiymon, wanbun}@kku.ac.th

Abstract—Antagonistic yeasts to Colletotrichum capsici

were isolated from rhizosphere soil, fruits and leaves of chili

plants. The majority of yeast isolates (60 isolates; 31.09%)

were isolated from rhizosphere soil. In dual culture tests,

five of the isolates screened (HS6, SS11, SLD5, SS10 and

PLN13) were found to inhibit C. capsici growth with

biocontrol efficacies as 43.12%, 42.50%, 41.87, 41.25 and

40.62%, respectively. Morphological characteristics of them

were examined by observing cell and colony patterns. They

produced septate pseudohyphae, holoblastic budding and

ascospore with two-layers. The colony of each antagonistic

yeast was globose, mucoid, white, glistering, raised and

smooth. Based on statistical analysis, these antagonistic

yeasts (HS6, SS11, SLD5, SS10 and PLN13) could

significantly control disease incidence in chili fruits when

compared control. The most effective antagonistic isolate

was PLN13, the percentage of survival in chili fruits being

60.00 %.

Index Terms—biological control, antagonistic yeast,

anthracnose disease, chili fruit

I. INTRODUCTION

Chili (Capsicum annuum L. var. acuminatum Fingerh.)

is one of the most important economic food crops in

Thailand for domestic usage and export. However, chili

cultivation is always infected by Colletotrichum capsici

causing Anthracnose, a serious disease in the plant [1].

Anthracnose destroys mature chili fruits during

cultivation, transportation and storage causing 50%

reduction of pre-and post-harvest in chili fruits [2]. One

way to control the disease is to use chemical substance

but this method can cause environmentally risky,

chemical resistant pathogens, and contamination in food.

Therefore, biological control can be an alternative way

for a disease management and so far several projects

have been reported that yeasts can be used as biological

agents against the plant pathogen in chili fruits. [3]. Our

objectives were to isolate yeasts from rhizosphere soil

and chili plant. In addition, the yeasts were tested the

efficiency to control anthracnose disease in vitro and in

vivo.

Manuscript received September 3 2013; revised November 17, 2013.

II. MATERIALS AND METHODS

A. The Fungal Pathogen, Colletotrichum capsici.

The pathogen, C. capsici, received from Department of

Plant Pathology, Faculty of Agriculture, Khon Kaen

University. This pathogen was cultured on Potato

dextrose agar (PDA) slant kept at 5oC until used.

B. Isolation of Yeast from Samples

Samples were collected from rhizosphere soil, fruit

and leaves of chili plants from agricultural field in the

Northeast region of Thailand. Ten grams of sample were

used for serial dilutions in sterile distilled water. After

that, 0.1 ml of each dilution was spread on yeast malt

extract agar (YM agar). The plates were incubated at 30

C for 48 h. Yeast colonies were examined under the

microscope and different morphological colonies were

selected. The yeast isolates were re-streaked on YM agar

to obtain pure cultures and they were maintained on YM

agar. The cultures were stored at 4 C until used [4].

C. Screening for Yeast Antagonists against C. capsici

Based on dual culture method, the 0.5 mm in diameter

of 10 days old C. capsici mycelial disc was put into the

center of the PDA plate. The plate was incubated at 30

C for 4 days. Next, the 48 h of isolated yeast was

streaked away from the pathogen 1 centimeter. Each

inoculation was done in four replicates. The inoculated

plates were incubated at 25 C for 10 days. The radius of

colony of the pathogen was measured compared with the

control [5]. The yeast isolates that inhibited fungal

growth called antagonistic yeasts were used for further

studies.

D. Morphological Characteristics of Antagonistic

Yeasts

Yeast antagonists were studies morphological

characteristics (texture, color, surface, elevation, margin,

cell arrangement and type of spore). The yeast cells were

cultivated on solid medium (YM agar). The cultures were

incubated for 48-72 h at 25 °C. Characteristics of cells

and colony patterns were observed according to the

method described by Kurtzman and Fell (1998). The

average length and width of 20 cells from each yeast

strain were measured.

Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013

2013 Engineering and Technology Publishing 201doi: 10.12720/jolst.1.4.201-204

,

E. Postharvest Disease Control by Antagonistic Yeasts

Each of five antagonistic yeasts (HS6, SS11, SLD5,

SS10 and PLN13) was cultured in YMB at 25 °C, 48h.

Cells and culture filtrate were collected by centrifugation

at 12000 rpm for 20 min. Intact chili fruits samples were

sterilized with 0.6 % sodium hypochlorite for 5 min,

washed with tap water, air drying and treated with 70%

ethanol. One group of 60 fruit samples was thoroughly

sprayed with 300 ml of cell suspension in distilled water

(5x108 cells/ml), another group was sprayed with culture

filtrate of each yeast strain. The samples were placed on

plastic boxes. The boxes were placed in the dark at 28 °C

for 2 h and followed by storage at 28 °C for 20 days. The

percentages of anthracnose survival of chili fruit in each

group were recorded at 5, 10, 15 and 20 days. Data

analysis were done by using one-Way ANOVA of IBM

SPSS statistics (version 19.0), SPSS Inc., Chicago, IL

USA). The least significant difference (LSD) test at

P<0.05 was made for mean comparision.

III. RESULTS

A. Isolation of Yeast from Samples

In primary screening, a total of 193 yeast isolates was

separated from rhizosphere soil, fruits and leaves of chili

plants. The majority of isolated yeast were found from

rhizosphere soil (60 isolates; 31.09%), followed by

normal leaves (47 isolates; 24.35%), disease leaves (32

isolates; 16.58%), disease fruits (31 isolates; 16.06%)

and normal fruits (23 isolates; 11.92%) (Fig. 1).

Figure. 1. Numbers and percentages of yeast isolates isolated from chili plantation.

B. Screening for Yeast Antagonists Against C. capsici

All yeast strains were selected for mycelia growth

inhibition of C. capsici in vitro test. The result showed

that 160 isolated yeasts (82.90%) effectively suppressed

to C. capsici while 33 isolated yeasts (17.10%) could not

inhibit. Five isolates (HS6, SS11, SLD5, SS10 and

PLN13) were able to inhibit the pathogen at 43.12%,

42.50%, 41.87, 41.25 and 40.62%, respectively. They

were not considered difference from the test (P>0.05) but

were different from the control treatment. These results

are shown in Table I. Thus, 5 isolated yeasts were tested

further to confirm their potentiality for controlling chili

anthracnose caused by C. capsici in vivo test.

TABLE I. RADIAL AVERAGES OF COLLETOTRICHUM CAPSICI

COLONIES AND PERCENT INHIBITION BY DUAL CULTURE TEST

Yeast isolates Radial average (mm) Inhibition (%)

HS6 23.25±0.50a 43.12±2.39a

SS11 23.00±1.15a 42.50±2.88a SLD5 23.25±3.30a 41.87±3.26a

SS10 23.50±1.73a 41.25±4.33a

PLN13 23.75±0.50a 40.62±1.25a

Control 40.00±0.00b 0.00±0.00b

P-value 0.079 0.083

% C.V. 1.73 2.54

Control group was uninoculated with yeast isolate.

Mean in the same column followed by a different letter indicate significant difference (P <0.05) according to LSD test.

C. Morphological Characteristics of Antagonistic

Yeasts

Morphological characteristics of antagonistic yeasts

were examined by observing cell and colony patterns

according to the method described by Kurtzman and Fell

(1998). Yeast isolates (HS6, SS11, SLD5, SS10 and

PLN13) showed similarly morphological characteristics

(Table II). All isolates could produce pseudohyphae

(septate hyphae), holoblastic budding and ascospore with

two-layered walls (Fig. 2). The colony patterns were

globose, mucoid, color of white, glistering, raised and

smooth (Fig. 3).

TABLE II. IDENTIFICATION OF ANTAGONISTIC YEASTS BY

MORPHOLOGICAL CHARACTERISTICS.

Yeast

isolate Texture Color Surface Elevation Margin

HS6 mucoid white glistering raised smooth

SS11 mucoid white glistering raised smooth

SLD5 mucoid white glistering raised smooth

SS10 mucoid white glistering raised smooth

PLN13 mucoid white glistering raised smooth

Yeast

isolate Arrangement

Size of

cell (µm) Pseudohyphae

Type of

spore

HS6 single 4.55±0.62 + ascospore

SS11 single 4.27±0.86 + ascospore

SLD5 single 4.90±0.55 + ascospore

SS10 single 4.67±0.71 + ascospore

PLN13 single 5.22±0.75 + ascospore

+ = produced pseudohyphae

Figure. 2. HS6 isolate showed pseudohyphae with septate hyphae (a), ascospore with two-layered walls and holoblastic budding (b), scale bar

= 1 µm.

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2013 Engineering and Technology Publishing 202

Figure. 3. Colony patterns of antagonistic yeasts; globose, mucoid,

white, glistering, raised and smooth, a=HS6, b=SS11, c=SLD5, d=SS10

and e=PLN13

D. Postharvest Disease Control by Yeast Antagonists

The chili fruits were sprayed with cell suspension and

culture filtrate of each isolate (HS6, SS11, SLD5, SS10

and PLN13). All isolates could control anthracnose in

chili fruits when compared with control. After 10 days of

treatment, isolates PLN13 and SS11 showed the highest

percentage of suvival of fruit from anthracnose at 60.00%

and 46.25 %

when treated chili fruits with cell

suspension and culture filtrate, respectively (Table III).

TABLE III. THE PERCENTAGE OF SUVIVAL OF CHILI FRUITS FROM THE

DISEASE (ANTHRACNOSE CAUSED BY C. CAPSICI)

Yeast

isolate

Survival of chili fruits from anthracnose (%) 5 days 10 days

Cell

suspension

Culture

filtrate

Cell

suspension

Culture

filtrate

HS6 100a 100a 53.75a 40.00d

SS11 100a 100a 42.50b 46.25bc

SLD5 100a 100a 17.50c 12.50d

SS10 100a 100a 56.25a 36.25a

PLN13 100a 100a 60.00a 38.75ab

Control 100a 100a 6.25d 0.00e

%CV - - 15.95 12.67

Yeast

isolate

Survival of chili fruits from anthracnose (%)

15 days 20 days

Cell

suspension

Culture

filtrate

Cell

suspension

Culture

filtrate

HS6 51.25a 36.25a 36.25a 21.25a

SS11 30.00c 37.50a 18.75b 23.75a

SLD5 17.50d 10.00b 11.25c 8.75b

SS10 43.75b 38.75a 25.00b 25.00a

PLN13 55.00a 38.75a 40.00a 21.25a

Control 0.00e 0.00c 0.00d 0.00c

%CV 11.83 10.44 18.64 18.32

The results are presented as mean of percentage. Value of each column followed by a different letter indicate significant differences (P<0.05)

according to LSD test.

IV. DISCUSSION

One hundred ninety three isolates of yeasts were

isolated from rhizosphere soil, fruits and leaves of chili

plants. In primary screening, the majority of yeast

isolates were found from rhizosphere soil because of

complex environment and high microbial diversity in

soils [1]. The 5 isolates of yeasts (HS6, SS11, SLD5,

SS10 and PLN13) were tested antagonists against

mycelia growth of C. capsici by dual culture method. The

HS6 isolate showed the highest inhibition (46.25%).

However, this inhibition is lower than previous study by

Chanchaichaovivat et al. [6] reported that the antagonistic

yeast (Candida quercitrusa isolate L2) to C. capsici was

isolated from chili showed control at 66.40%. The

antagonistic yeasts have been studied morphological

characteristics. The colony patterns were reported

according to Table 2. The cells are golbose, ascospore

with two-layered walls, pesudohyphae with septate

hyphae and holoblastic budding (Fig. 2) and sizes of cells

is range from 4.27 to 5.22 µm. These characteristics were

identified as Candida species [7]. However, the results of

identification will be confirmed by molecular technique.

Based on postharvest disease control by antagonistic

yeasts, PLN13 isolate showed high efficacy to control

anthracnose both treated with cell suspension and culture

filtrate. This study suggests the yeast strain HS6, SS11,

SLD5, SS10 and PLN13 as an alternative to anthracnose

disease control by C. capsici in postharvest chili fruits.

Nevertheless, more studies including greenhouse and

field need to be performed.

ACKNOWLEDGEMENTS

This work was supported with grants of Rajamangala

University of Technology Isan, Kalasin Campus.

REFERENCE

[1] A. Chanchaichaovivat, “Using hand on Yeast biological control for colletotrichum capsici to teach organism interrelationship

concepts and encourage critical thinking,” The degree of Doctor

of Philosophy (Science and Technology), Faculty of Graduates studies, Mahidol University, 2008.

[2] U. A. Druvefors, “Yeast biocontrol of grain spoilage moulds:

Mode of action of pichia anomala,” Doctoral thesis, Swedish University of Agriculture Science, Uppsala, 2004.

[3] W. J. Janisiewicz, B. Leverentz, W. S. Conway, R. A. Saftner, A.

N. Reed, and M. J. Camp, “Control of bitter rot and blue mold of

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2013 Engineering and Technology Publishing 203

apples by intrgrating heat and antagonist treatments on 1-MCP

treated fruit stored under controlled atmosphere conditions,”

Postharvest Biology and Technology, vol. 29, pp. 129-143, 2003.

[4] C. P. Kurtzman and J. W. Fell. The Yeast: A Taxonomic Study. 4th edition, ELSEVIER, 1998, pp.1035.

[5] P. Plodpai. “Biocontrol of some chilli fungus disease by bacillus

spp,” Master of Science in Plant Pathology, Prince of Songkla University, Songkla, 2007, pp. 104.

[6] A. Chanchaichaovivat, C. R. Pintip, and P, Bhinyo, “Screening

and identification of yeast strains from fruits and vegetables: Potential for biological control of postharvest chili anthracnose

(Colletotrichum capsici),” Biological Control, vol. 42, pp. 326-

335, 2007. [7] C. P. Kurtzman and J. W. Fell, The Yeast: A Taxonomic Study, 5th

edition. ELSEVIER, 2011, vol. 2, pp. 1103-1144.

Miss Punika Chaisemsaeng graduated Bachelor

and Master degree from Biology Department,

Faculty of Science, Khon Kaen University,

Thailand. She is a Ph. D. student at Microbiology Department, Faculty of Science, Khon Kaen

University, Thailand. The research area is

biological control and their application.

Wiyada Mongkolthanaruk graduated from Sheffield University in

molecular biology and biotechnology. The research areas are

endophytic bacteria and their applications, bacteria enzyme for biotechnology, e.g. laccase, lipase. Also, she is interested in bioactive

compounds for plant-microbe interactions.

Wandee Bunyarachata is Doctor of Philosophy (Plant Pathology),

Khon Kaen University, Thailand. (Thesis: Race Identification of

Fusarium oxysporum f. sp. lycopersici, Wilt Pathogen of Tomato, and Efficacy of Soil Fungi Controlling the Disease). He got Master of

Science (Industrial Microbiology), Chulalongkorn University, Thailand.

Bachelor of Science (Biology), Chiang Mai Univrtsity, Thailand. The research areas are biological control using microorganisms, microbial

enzyme (cellulase), and bioactive compound from microorganisms.

Journal of Life Sciences and Technologies Vol. 1, No. 4, December 2013

2013 Engineering and Technology Publishing 204