Exploration of Yeast and Bacteria as Antagonist Agents

Candidates of Citrus Foot Rot Disease

(Botryodiplodia theobromae (Pat.)
Nur Annisa Shalehah
A34120104

Supervised by:
Prof Dr Ir Meity Suradji Sinaga, MSc

DEPARTMENT OF PLANT PROTECTION

FACULTY OF AGRICULTURE
BOGOR AGRICULTURAL UNIVERSITY
2016
INTRODUCTION

Citrus is highly demanded by the people in Indonesia

Table 1 Production, harvested area, and productivity of citrus in Indonesia

Year
Production, harvest area,
and productivity
2011 2012 2013 2014 2015

Production (ton) 1 721 880 1 498 394 1 548 394 1 785 256 1 744 330

Harvested area (ha) 47 181 46 187 48 154 51 098 48 119

Productivity (ton/ha) 36.50 32.44 32.15 34.97 33.92

Source: Ministry of Agriculture (2016)

INTRODUCTION

• One of the main disease

that attacks citrus
plantation in Indonesia
was stem-end rot disease.
• Sinaga et al. (2009) have
found that the main
causative agent of stem
end rot disease in 11 citrus
central plantation area in
Indonesia was
Botryodiplodia
Symptoms of foot rot disease on citrus theobromae.
INTRODUCTION

Previous studies about the effectiveness of biocontrol agents

to control B. theobromae (Retnosari 2011, Supraba 2014,
Hardiati 2015).

Some yeast can growth rapidly, produce antibiotics and

cellwall degrading enzymes, induce plant host resistance, and
produce plant growth controller (Suprapta)

The aims of this study was to isolate yeast and or bacteria as

biocontrol agent of B. theobromae in vitro.
MATERIALS AND METHODS
This research was conducted in Laboratory of Plant Mycology,
Department of Plant Protection from April until August 2016.

Materials

Blood agar Colloidal chitin

Citrus bark, B. theobromae Tobacco plant media media
root, and leaves isolate

Yeast Glucose
Nutrient Agar Malt Extract Nutrient Broth
Potato Dextrose Chloramphenicol
(NA) Agar (MEA) (NB)
Agar (PDA) Agar (YGCA)
MATERIALS AND METHODS
Equipments

Scale Shaker Mortar and pistil

Light microscope Compound microscope

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METHODS OVERVIEW

Isolation of
epiphytic and Characterization
endophytic yeast of microbial
and bacteria colonies

Pathogenicity
In vitro assay
test
METHODS
Isolation of epiphytic yeast and bacteria

0.1 ml to
Serial NA, MEA,
10 g bark, 100 ml
120 rpm, dilution YGCA
root, or sterile
15 minutes from 10-1
leaves water Incubate
until 10-3
for 2 to 3
days
METHODS
Isolation of endophytic yeast and bacteria

10 g bark, 0.1 ml to
root, or Serial NA, MEA,
100 ml Crushed
leaves dilution YGCA
sterile with
from 10-1
Surface water mortar Incubate
until 10-3
sterilization for 2 to 3
days
METHODS
Colony observation and isolates preservation

NA, MEA, or YGCA PDA or NA media PDA or NA media

METHODS

Yeast pathogenicity test

Counting the
number of yeast Inoculation of
Sterilization of
yeast suspension
young citrus stem cells with to stem
methods

Bacteria hypersensitivity test

Bacteria isolates Injection of

were shaken in bacteria
Observation
NB for 24h at suspension to
120 RPM tobacco leaf

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METHODS
Hyperparasitism assay

Yeast
B. theobromae

WA media
Incubated for 7 days

Affinities of yeast Number of yeast cell

that adhere to hyphae
- 0
Sparse attachment <10
Loose attachment 10-50
Heavy attachment >51
Chan and Tian (2005)
 METHODS
Dual culture assay

Yeast or bacteria isolate

B. theobromae

3 cm 3 cm

𝐑𝟏 −𝐑𝟐
RIR = X 100%
𝐑𝟏

RIR = Relative inhibition rate

R1 = Radius of the radial growth of the
pathogen in control plate
PDA media
Incubated for 5 days R2 = Radius of the radial growth of the
pathogen towards the opponent
Wisniewski et al. (2007) antagonist in test plate
 METHODS
Volatile compound production assay

Yeast or bacteria
B. theobromae

Incubated for 7 days

𝐃𝟏 −𝐃𝟐
RIR = X 100%
𝐃𝟏

RIR = Relative inhibition rate

D1 = Diameter of radial growth of the
pathogen in control
D2 = Diameter of radial growth of the
pathogen in treatment
Jeyaseelan et al. (2012)
 METHODS
METHODS
Chitinase production assay

ΔY = Y2/Y1
ΔY = Chitinolitic index
Choloidal chitin Y2 = Diameter of yeast
media colony
Y1 = Diameter of clear zone
around yeast colony

Clear zone
Yeast or bacteria suspension

Hartati et al. (2014)

 METHODS
METHODS
Yeast and bacteria safety test against mammals

Blood agar media

Darkened zone

Clear zone
Yeast or bacteria suspension

Manns et al. (19944)

 METHODS
METHODS
Experimental Design and Data Analysis

All of in vitro assays were conducted using completely

randomized design with 3 replications. Data from dual
culture assay and volatile compound assay were analyzed
and tested with Duncan’s Multiple Range test with α = 5%
using software SAS 9.1.

Manns et al. (19944)

RESULT AND DISCUSSION
Table 1 Result of isolation from citrus plants
Isolate Group Source Type
MAFL001 Yeast Root Epiphytic
MBFG001 Yeast Bark Epiphytic
MBFG002 Yeast Bark Epiphytic
MCFG002 Yeast Leaf Epiphytic
MCFG003 Yeast Leaf Epiphytic
MCFG004 Yeast Leaf Epiphytic
TBDL003 Bacteria Bark Endophytic
TBFG001 Bacteria Bark Epiphytic
TBFG004 Bacteria Bark Epiphytic
TBFG005 Bacteria Bark Epiphytic
TBFG006 Bacteria Bark Epiphytic
TBFG007 Bacteria Bark Epiphytic
TBFG008 Bacteria Bark Epiphytic
TBFG010 Bacteria Bark Epiphytic
TCFG006 Bacteria Leaf Epiphytic
TCFG011 Bacteria Leaf Epiphytic
23/01/2017 TCFG012 Bacteria Leaf Epiphytic
RESULT AND DISCUSSION
Table 2 Characteristics of yeasts colony

Isolate Form Color Margin Elevation

MAFL001 Circular Milky white Entire Corvex

MBFG001 Circular Milky white Undulate Corvex

MBFG002 Circular Pale pink Entire Corvex

Circular with
MCFG002 Light brown Entire Raised
raised margin
Circular with
MCFG003 Pink Entire Corvex
raised margin
MCFG004 Irregular Pink Undulate Raised

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RESULT AND DISCUSSION

Fig 1 Yeast cells characteristics at magnificent 100x10: Isolate

MAFL001 (a), MBFG001 (b), MBFG002 (c), MCFG002 (d),
MCFG003 (e), and MCFG004 (f)

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RESULT AND DISCUSSION
Table 3 Characteristics of bacteria colony
Isolate Form Color Margin Elevation
TBDL003 Circular Milky white Curled Convex
TBFG001 Circular Pink Curled Convex
TBFG004 Irregular Reddish Lobate Raised
TBFG005 Circular Pink Curled Raised
TBFG006 Circular White Entire Convex

TBFG007 Circular White Entire Convex

TBFG008 Circular Yellowish Entire Flat
TBFG010 Circular Pink Entire Umbonate
TCFG006 Circular Pink Undulate Convex
TCFG011 Irregular Pink Lobate Umbonate
TCFG012 Circular Milky white Entire Convex

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RESULT AND DISCUSSION
Pathogenicity test of yeast isolates
Table 4 Result of pathogenicity test of yeast isolates on young citrus stem and blood agar

Media
Isolate
Young citrus stema Blood agarb
MAFL001 ++ -
MBFG001 ++ -
MBFG002 ++ -
MCFG002 ++ -
MCFG003 ++ -
MCFG004 ++ -
a (-) = necrotic symptom on stem; (+) = no necrotic symptom on stem; (++) = stem was colonize
by yeast
b (+) = clear zone or darkened zone on blood agar media; (-) = no clear zone or darkened zone

on blood agar media

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RESULT AND DISCUSSION
Pathogenicity test of yeast isolates

a b c d e f g

Fig 2 Colonization of young citrus stem by yeast isolate MAFL001 (a), MBFG001 (b),
MBFG002 (c), MCFG002 (d), MCFG003 (e), MCFG004 (f), and necrotic
symptom on positive control (g)

Yeast ability to take nutrients and adaptate well with plant environment
without causing negative effect to plant

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RESULT AND DISCUSSION
Pathogenicity test of bacteria isolates
Table 5 Result of pathogenicity test of bacteria isolates on tobacco leaf and blood agar

Media
Isolate
Tobacco leafa Blood agarb
TBDL003 - -
TBFG001 - -
TBFG004 - -
TBFG005 + -
TBFG006 - -
TBFG007 - -
TBFG008 - -
TBFG010 - -
TCFG006 - -
TCFG011 - -
TCFG012 + symptom on leaf
a (+) = necrotic symptom on leaf; (-) = no necrotic -
b (+) = clear zone or darkened zone on blood agar media; (-) = no clear zone or darkened zone
on blood agar media
RESULT AND DISCUSSION
Pathogenicity test of bacteria isolates

a b c d

e f g h

i j k l

Fig 3 Sign of chlorotic symptom on tobacco leaf after inoculated with bacteria isolate
TBFG005 (d) and isolate TCFG001 (k), and necrotic symptom on positive control (l)

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RESULT AND DISCUSSION
Yeast efficacy against B. theobromae in vitro
Table 6 Result of antagonistic ability of yeast isolates against B. theobromae in vitro
RIR in dual
RIR by volatile Chitinolitic Hyperpara
Isolate culture Effectivityc
compound (%)a index sitismb
assay(%)a
Control 0.00 0.00 0.00 - -
MAFL001 19.22a 15.09a 0.00 +++ ++
MBFG001 0.00b 7.08b 0.00 ++ -
MBFG002 0.78b 12.74ab 0.00 +++ +
MCFG002 3.92b 10.38ab 0.00 - +
MCFG003 4.71b 16.04a 3.12 +++ ++
MCFG004 0.00b 7.08b 0.00 +++ +
a Number followed by different alphabet in the same column shows real difference at DMRT α=5
b(-) Didn’t show hyperparasitism activity; (+) Low hyperparasitism activity; (++) Medium hyperparasitism
activity; (+++)) High hyperparasitism activity
c(-) Not effective (+) Low effectivity; (++) Medium effectivity; (+++) High effectivity

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RESULT AND DISCUSSION
Yeast efficacy against B. theobromae in vitro
Volatile compound production assay

a b
Fig 4 Mycellial growth of B. theobrmae was suppressed and became thinner in
treatment with yeast isolate MAFL001 (a) compared to control (b) 4 days after
inoculation
Volatile compounds that were produced by yeast in phyllum Ascomycota that were
isolated from tropical area were found in the form of alcohol, aldehid, and esther
(Buzzini et al. 2003)
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RESULT AND DISCUSSION
Yeast efficacy against B. theobromae in vitro

a b

MAFL001 MBFG001

c d g

MBFG002 MCFG002 Kontrol

e f
MCFG003 MCFG004

Fig 5 Difference between colony color of B. theobromae in yeast treatment (a-f) to

control that had darkened considerably (b) 7 days after inoculation
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RESULT AND DISCUSSION
Yeast efficacy against B. theobromae in vitro

Chitinase production assay

a b

Fig 6 Clear zone around yeast isolate MCFG004 (a) and no sign of clear zone around
yeast isolate MCFG004 (b) 7 days after inoculation

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RESULT AND DISCUSSION
Yeast efficacy against B. theobromae in vitro

Dual culture assay

a b c d

e f g

Fig 7 Mycellium of B. theobromae looked thinner in treatment with yeast isolates

(a-f) compared to control (g)
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RESULT AND DISCUSSION
Yeast efficacy against B. theobromae in vitro

Hyperparasitism assay

a b c d

Fig 8 Red arrows show malformation hyphae of B. theobromae after parasitized by

yeast isolate MAFL001 (a), MBFG002 (b), MCFG003 (c), and MCFG004 (d)

Production of cell wall degrading enzymes like chitinase and β-1,3-glucanase were
correlated with yeast cells ability to adhere to pathogen hyphae (Chan & Tian 2005)

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RESULT AND DISCUSSION
Bacteria efficacy against B. theobromae in vitro
Table 7 Result of antagonistic ability of bacteria isolates against B. theobromae in vitro
RIR in dual
RIR by volatile Chitinolitic
Isolate culture Effectivityb
compound (%)a index
assay(%)a
Control 0.00 0.00 0.00 -
TBDL003 41.79ab 1.69cd 0.00 +
TBFG001 -13.59b -8.61d 0.00 -
TBFG004 20.00ab 14.79c 0.00 +
TBFG006 52.82ab 16.67c 0.00 ++
TBFG007 48.72ab 40.54ab 0.00 ++
TBFG008 58.97a 19.94bc 1.87 ++
TBFG010 -2.56ab 15.73c 0.00 -
TCFG006 8.72ab 7.77cd 0.00 -
TCFG011 12.82ab 42.42a 0.00 ++
a Number followed by different alphabet in the same column shows real difference at DMRT α=5
b(-) Not effective (+) Low effectivity; (++) Medium effectivity; (+++) High effectivity.
RESULT AND DISCUSSION
Bacteria efficacy against B. theobromae in vitro

Volatile compound production assay

a b c d

Fig 9 No sign of mycellial growth of B. theobrmae in treatment with bacteria isolate

TBFG008 (a), and slow mycellial growth of B. theobromae in treatment with
bacteria isolate TBFG006 (b) and TBFG007 (c) compared to control (d)

Bacteria could produce many volatile organic compounds as a mixture of alcohol,

aldehide, ester, terpenoid, and other light molecules that can evaporate easily
(Bennett et al. 2012)
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RESULT AND DISCUSSION
Bacteria efficacy against B. theobromae in vitro

Dual culture assay

a b c

Fig 10 Clear zone at the margin of bacteria isolate TBFG007 and the edge of
B. theobromae mycellia(a), thinning of B. theobromae mycellia near bacteria
isolate TCFG011 (b) and control (c)

Production of antifungal compounds occured when fungi and bacteria are within a
close range or if there is physical contact among
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RESULT AND DISCUSSION
Bacteria efficacy against B. theobromae in vitro

Chitinase production assay

Fig 11 Clear zone around bacteria isolate TBFG008

Chitinase was produced by yeast or bacteria by hydrolizing chitin to N-asetil

glucosamina monomer and was used as carbon source (Yanai et al. 1992)

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 CONCLUSION
Conclusion
• The result from isolation of citrus plant were six nonpathogenic epiphytic yeast,
eight nonpathogenic epiphytic bacteria, one nonpathogenic endophytic bacteria,
and two plant pathogenic epiphytic bacteria.
• Yeast isolates MAFL001 and MCFG003 could inhibit B. theobromae growth
directly and had high hyperparasitism activity. Isolate MAFL001 could also
produce toxic volatile compound while isolate MCFG003 could produce chitinase.
• Bacteria isolates TBFG007 and TBFG008 could inhibit B. theobromae growth
directly and produce volatil compound and could produce chitinase for isolate
TBFG008.
• These four isolates were showing the best potential as biocontrol agents of B.
theobromae in vitro.

Suggestion
In vivo and in planta assay need to be conducted to find out yeasts and bacteria
potency in suppressing disease incidence and disease severity of stem-end rot disease

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THANK YOU