3. Suryo Wiyono.cdr

BIOTROPIA Vol. 20 No. 1, 2013: 19 - 28

POWDER FORMULATION OF ANTAGONISTIC
YEASTS, AND

AS BIOFUNGICIDES
CRYPTOCOCCUS ALBIDUS

CRYPTOCOCCUS TERREUS

SURYO WIYONO* and WIDODO

Recipient of BIOTROP Research Grant 2009/Accepted 14 May 2012

This study aimed 1) to investigate the compatibility of yeast antagonists i.e. and
2) to obtain suitable carrier in powder formulation for those two yeasts, 3) to get

appropriate formulation additives for those yeasts, and 4) to obtain optimum powder
formulation consisting of yeasts, carrier materials and additives. Compatibility of and

were tested through bio-assay against on detached banana fruit
and on detached tomato leaves. Compatibility was indicated by no reduction of
antagonistic activity. Benomyl-resistant mutant of and cycloheximide-resistant mutant
of were mixed with sterilized tapioca, talc and kaolin to get initial yeasts density of 8.5
log cfu/g and water content of 15%, then packed in plastic bag and stored under room
temperature. Survival of formulated yeasts was assessed monthly by planting on PDA medium
containing 150 ppm cycloheximide for , and 150 ppm benomyl for . Yeasts
population was expressed in log cfu/g materials. To determine the effect of carrier materials on
antagonistic activity, io-assay of formulated yeasts against pathogens was conducted after 3
months of storage. Tested additives CaCl , pure chitin and crab shell powder were added into
suspension of and to get concentration of 1.25%, 0.5 % and 0.1% (w/v).
Sterilized distilled water and yeasts without additives was used as control. Then, the treatments
were examined for the antagonistic activity through bio-asaay on detached banana fruits and
tomato leaves. Appropriate additive(s) was determined by its ability to increase antagonistic
activity of yeasts. Storability and antagonistic activity of and in the mixture
of best carrier and additive were examined. was compatible to .
Talc was the best carrier material supporting highest survival of , without
contamination of other microorganisms. Talc was the best carrier material for
formulation by maintaining its survival for six months of storage. Additives able to increase
antagonistic activity of were CaCl 0.1 %, pure chitin 0.1 % and crab shell powder
0.5%. All of tested additives materials did not affect antagonistic activity of
Supplementation of chitin and crab shell, both at the rate of 1.25% into talc-base powder
formulation increased survival of and .

formulation, yeast antagonists carrier agent,
talc, tapioca, kaolin, CaCl , chitin, crab shell powder

1
Department of Plant Protection, Faculty of Agriculture, Bogor Agricultural University

Darmaga, Bogor-Indonesia

C. albidus C.
terreus,

C. albidus
C. terreus Lasiodiplodia theobromae

Alternaria solani
C. albidus

C. terreus

C. terreus C. albidus

b
i.e.

C. albidus C. terreus

C. albidus C. terreus
Cryptococcus terreus C. albidus

C. terreus
C. albidus

C. terreus

C. albidus.

C. terreus C. albidus

, Cryptococcus terreus, Cryptococcus albidus,

ABSTRACT

Key words:

* Corresponding author : suryow@hotmail.com

INTRODUCTION

A group of novel promising biocontrol agents of plant disease are antagonistic
yeasts. The advantage of yeast as biocontrol agent is dry and heat tolerance, therefore,
adaptable on leaves and other aerial plant parts. It is fast growing, easy to be mass
cultured and socially more acceptable (Spadaro 2003).

Research in foreign countries showed the effectiveness of yeast in controlling plant
disease. A yeast antagonist was reported effective to control
apple rot disease caused by (Spadaro 2003). Moreover, near harvest
application of effectively control post harvest disease of
grapefruit (Karabulut . 2003). on tomato plants was reported to be
effectively controlled by the use of epiphytic yeasts strains 101
and US 7 and (Saligkarias . 2002).

and collected by the authors have been
screened for their antagonistic activity , bio-assay , field test and partially
characterized. and were effective yeast
antagonists against stem end rot of mango caused by and petal
blight of orchid caused by , respectively (Wiyono 2008;
Sugiprihatini 2009). Recently, the second yeast is also effective tested against leaf
blight of tomato caused by and chrysanthemum white rust caused by

. In addition, Fan and Tian (2001) reported that is also
effective in controlling apple post harvest diseases i.e. grey mold and blue mold.

After obtaining superior isolates of antagonists, technology for mass production
and formulation is required to develop an antagonist as biocontrol agent on
commercial scale. Since those two yeasts are relatively easy to be mass cultured in a
cheap medium (potato dextrose broth PDB), formulation technology is a critical step.
Suitable formulation technology is the main limiting factors in developing microbial
pesticides in Indonesia, even high numbers of effective antagonists had been isolated
(Santoso . 2005). Appropriate formulation technology will facilitate storage,
transportation, application technique and also bio-performance of antagonists. Solid
formulation is chosen to be developed for easier transportation and handling.

Formulation of beneficial microbes contains microbes, carrier and additives.
Combination of microbes will broaden the target of bio-fungicide. Optimization of
whole components ensures the quality of formulation and further commercial
application. Some minerals such as kaolin and talc can be used as carrier in formulation
of and (Kinay & Yildiz 2008). Additives
play also an important role in biocontrol performance. Calcium chloride and chitin
were used as additive enhancing antagonistic activity of yeasts antagonists

and . Compatibility of the two
yeast antagonists, appropriate as carrier materials and additives for and

has not been investigated yet.
The objectives of the research were: 1) to assess the compatibility of two yeast

antagonists and , 2)to obtain cheap material as carrier agent which
ensure long time survival and bio-performance of and 3) to get
formulation additives supporting storability and bioperformance of yeast antagonist

Metschnikowia pulcherrima
Penicillium

Metschnikowia fructicola
et al Botrytis cinerea

Candida guilliermondii
Candida oleophila et al

Cryptococcus albidus Cryptococcus terreus
in vitro in vivo

Cryptococcus albidus Cryptococcus terreus
Lasiodiplodia theobromae

Curvularia pallescens

Alternaria solani
Puccinia horiana Crypococcus albidus

et al

Metschnikowia pulcherrima Pichia guilliermondii

Candida
guilliermondii, Pichia membranefaciens, Cyptococcus laurentii

C.albidus C.
terreus

C. albidus C. terreus
C. albidus C. terreus,

BIOTROPIA Vol. 20 No. 1, 2013

C. albidus C. terreus

Cryptococcus albidus C. terreus
C. albidus

C. terreus
Lasiodiplodia theobromae

Alternaria solani

Lasiodiplodia theobromae

Alternaria. solani

C. albidus
C. terreus

L. theobromae A. solani

C albidus, C. terreus C albidus C. terreus

C. albidus C.
terreus

and ., and 4) to obtain optimum solid formulation in combination
with yeasts, carrier material and additives

Antagonistic yeasts used were and obtained from the
author's collection. For formulation purpose, benomyl-resistant mutant of
which is similar to antagonistic activity was generated. In addition, resistant to
cycloheximide was used in formulation experiment. and

collections of the author were used for bio assay of yeasts. Both of the two
yeast antagonists were cultured in potato dextrose broth (PDB Difco) and harvested at
early stationary phase of growth, centrifuged at 5000 g (Jouan Centrifuge BR4i), washed
with sterilized distilled water, mixed with saline solution adjusted to appropriate
density. was cultured on PDA (Difco) pH 5.5 for seven days and
the conidia harvested by soaking water on the surface and filtered with cheese cloth.

was cultured on S-medium and incubated for seven days under NUV
exposure to induce sporulation (Abadi 1987), prior to harvesting of conidia

io-assay of yeast antagonists was conducted based on previous technique
developed by the authors using detached banana fruits and detached leaves of tomato.
The detached organs were placed on moistened plastic pans (30 cm x 25 cm x 5 cm),
one plastic pan contained five banana fruits or tomato leaves. Detached banana fruits
were dipped in cell suspension of at 7 log cfu /mL, while the detached
leaves were dipped in cell suspension of 7 log cfu/mL, both added with
wetting agent Tween 20, 0.005 %. Then the treated banana fruits and tomato leaves
were air dried. Sterilized distilled water was used as control. Conidia suspension at 50 µl
of and , both at the density of 10 conidia /mL was placed on
the surface of fruit and leaves, respectively. Inoculated fruits and leaves were stored
under dark condition for 24 hours and then incubated under room temperature (27 C)
and photoperiods of 12: 12 (D:L). Disease severity was assessed by estimating of
necrosis part at five days after inoculation and expressed in percent. Effective
treatment was indicated by low disease severity

he treatment consists of , a mixture of and ,
and untreated (water). Each treatment was replicated five times. Yeast concentration
used for compatibility test was 5 x 10 cfu/mL for and 5 x 10 cfu/mL

, therefore obtaining final concentration of 7 log cfu/mL, with ratio of 1:1, and
added with wetting agent Tween 20, 0.005 %. Bio-assay was conducted on detached
fruit of banana and detached leaves of tomato (see bio-assay of antagonistic activity).

MATERIALS AND METHODS

Yeast and pathogen preparation

Bio-assay of antagonistic activity of yeasts

Compatibility of and

6 6

C. albidus C. terreus

Powder Formulation of Antagonistic Yeasts as Biofungicides Suryo Wiyono– et al.

If the two yeasts are compatible, it will be further tested in a formulation
experiment

aterials tested for carrier were kaolin powder, talc powder and tapioca. Each
material was regarded as treatment and replicated five times. The materials were
sterilized by standard autoclaving. Suspension of two yeast antagonists and its mixture
( preparation mentioned above) was mixed by spraying (hand sprayer Yoto 1-L, made
in Indonesia) yeast suspension on the tested materials in running blender, then air
dried to get final yeast density of 8.5 log cfu/g with 15% of water content of
formulation. Materials containing yeast were then packed in plastic bags and stored
under room temperature for 6 months. Each material containing yeast was assessed
for yeast survival and antagonistic activity every 30 days for six months. Survival of
yeast in each carrier materials was determined by plating on PDA (Difco) pH 5.5
containing 150 ppm benomyl for , and 150 ppm cycloheximide for
Colony isolated from each carrier material of three month storage was then tested for
its antagonistic activity using the technique as described in bioassay of antagonistic
activity

aterials screened for additives are pure chitin (Sigma), natural material containing
chitin i.e. crab shell powder, and calcium chloride. Methods for testing the formulation
additives are based on previously developed technique (Wiyono . 2008). Each
material was tested with water-based suspension/solution at the rate of 1.25 %; 0.5%;
0.1% (w/v). Suspension of and at density of 7 log cfu/mL mixed
with the tested additives and final concentration of additives was adjusted to the tested
concentration. Yeast suspension without addition of tested materials was used as
control. Yeasts with various additives treatment was furthermore tested for their
antagonistic activity using the technique as described in bioassay of antagonistic
activity

est results obtained from previous experiment with yeasts compatibility, carrier
materials and additives were then continued to test for suitable powder formulation .
Combination of yeasts, selected carrier materials, selected additives in powder
formulation was conducted. The survival and antagonistic activity were also assessed
every 30 days for four months

he research showed that was compatible to . Effectiveness

Screening for carrier materials

Screening for additives

Combination of suitable yeast composition, carrier materials and additives

Compatibility of dan

C. albidus C. terreus.

et al

C. albidus C. terreus

C. terrreus C. albidus

RESULTS AND DISCUSSION

C. terreus C. albidus

BIOTROPIA Vol. 20 No. 1, 2013

of the two yeast antagonists did not decrease in mixed application (Tables 1 and 2).
The compatibility of the two yeasts make broader spectrum of the mixture. One
important advantage of antagonists combination is the possibility to obtain a broader
spectrum (Burges & Jones 1998).

Survival of dan in various carrier materialsC. terreus C. albidus

.
E

.
T

arrier materials are important component of formulation for maintaining
microbes survival and antagonistic activity. Among materials tested for ,
tapioca provided the highest survival. Overall, could survive in all tested
carriers for four months. During five months of storage could not be
detected anymore in all of the tested materials (Table3). Talc powder provided highest
survival for (Table 4). Up to six months of storage, talc powder still resulted
in relatively high survival (4.60 log cfu/g)

ven though tapioca is the best material for storage, it has high
contamination level of other fungi. Tapioca is an organic flour, hence it can act as
nutrition for some other fungi and bacteria. This was not the case with mineral powder
such as kaolin and talc. Kinay and Yildiz (2007) stated that talc in granular formulation
is able to provide storability of antagonistic yeasts and

for more than 6 months. Storability of talc was better than kaolin.
Storability of talc in this experiment was lower than that reported by Kinay and Yildiz
(2007), because this experiment used powder formulation instead of granular form

he use of talc as carrier material in powder formulation is also able to maintain
bio-performance of the two yeasts in storage (Tables 5 and 6). Antagonistic activity of

C. terreus
C. terreus

C. terreus

C. albidus
C. albidus

C. terreus

Metschnikowia pulcherrima Pichia
guilliermondii

Table 1. Compatibility of and in controlling fruit rot of banana
caused by

Cryptococcus albidus C. terreus
L. theobromae

Treatment Disease severity (%)

Untreated 34.53 b

Cryptococcus albidus 26.00ab

Cryptococcus terreus

C. terreus+ C. albidus 7.53 a

Note:
Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

Table 2. Compatibility of and in controlling alternaria leaf blight
of tomato caused by

Cryptococcus albidus C. terreus
Alternaria solani

Treatment Disease severity (%)

Untreated 10.58 b

Cryptococcus terreus 5.55 a

C. terreus+ C. albidus 5.48 a

Note:
Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

Powder Formulation of Antagonistic Yeasts as Biofungicides Suryo Wiyono– et al.

BIOTROPIA Vol. 20 No. 1, 2013

Table 3. Survival of on various carrier materials in powder formulationC. terreus

Carrier

Materials

Yeast density (log cfu/g) in i-th month

0 1 2 3 4

Tapioca 8.70 a 7.06 b 7.23 b 7.36 b 4.18 b

Talc 8.65 a 6.00 a 6.54 a 5.69 a 4.50 b

Kaolin 8.65 a 6.67 a 6.24 a 6.26 a 3.82 a

Note:

- = undetected
Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

Table 4. Survival of on various materials in powder formulationC. albidus

Carrier

Materials

Yeast density (log cfu/g) in i-th month

0 1 2 3 4 5 6

Tapioca 8.24 a 5.95 a 6.98 a 4.21 a - - -

Talc 8.72 a 5.70 a 6.65 a 5.98 b 6.17 4.20 4.65

Kaolin 8.60 a 5.25 a - - - - -

Note:

-= Undetected
Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

Table 5. Antagonistic activity of formulated with different carrier materials against
alternaria leaf blight of tomato (after 3 months storage)

C. terreus

Carrier Materials Alternaria leaf blight severity (%)

Control (water) 7.64 b

Unformulated fresh yeast 2.13 a

Tapioca 0 a

Talc 2.12 a

Kaolin 1.13a

Note:

Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

Table 6. Antagonistic activity of formulated with different carrier materials against
fruit rot of banana (after 3 month storage)

C. albidus

Carrier Materials Fruit rot severity

(%)

Control (water) 16.23 b

Unformulated fresh yeast 5.24 a

Tapioca 9.14 a

Talc 5.22 a

Kaolin -

Note:
Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

C. albidus C. terreus

Pseudomonas fluorescens et al

C. terreus

C. albidus,

and in all tested materials (except kaolin) did not decrease after 3
months of storage indicated by no significant difference to unformulated fresh yeast

ormulation additives such as additional nutrients are often important in
formulation of beneficial microbes. Some formulation additives are able to increase
antagonistic activity of antagonistic microbes, for example zinc and manganese can
improve antagonistic activity of B5 (Wiyono . 2008). Among
tested additives, Ca Cl 0.1%, pure chitin 0.1% and crab shell powder 0.5% significantly
increase antagonistic activity of (Table 7). Even though there was no
significant difference among the three treatments, crab shell powder provided highest
increase of antagonistic activity

dditives treatment did not increase significantly antagonistic activity of
however CaCl 0.5%, crab shell powder 1.25% and pure chitin 1.25% tend to increase

.
A

Additives-mediated enhancement of antagonistic activity of and
.

C. terreus C.
albidus

Table 7. Effect of additives on the antagonistic activity of C. terreus

Treatment Conc. (% w/v) Alternaria leaf blight severity (%)

Water 25.45 e
Yeast without additives 9.52 cd
Ca Cl2 1.25 7.12 abcd
Ca Cl2 0.5 5.35 abc
Ca Cl2 0.1 4.52 ab
Chitin 1.25 10.00 d
Chitin 0.5 8.35 bcd

Chitin 0.1 4.25 ab
Crab shell powder 1.25 4.80 ab
Crab shell powder 0.5 3.14 a
Crab shell powder 0.1 3.94 ab

Note:

Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

Table 8. Effect of additives on the antagonistic activity of C. albidus

Treatment Conc. (% w/v) Fruit rot severity (%)

Water 55.00 c

Yeast without additives 35.00 ab

Ca Cl2 1.25 41.25 ab

Ca Cl2 0.5 21.25 a

Ca Cl2 0.1 43.75 ab

Chitin 1.25 15.75 a

Chitin 0.5 53.75 b

Chitin 0.1 43.75 ab

Crab shell powder 1.25 21.25 a

Crab shell powder 0.5 41.25 ab

Crab shell powder 0.1 43.75 ab
Note:
Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

Powder Formulation of Antagonistic Yeasts as Biofungicides Suryo Wiyono– et al.

BIOTROPIA Vol. 20 No. 1, 2013

antagonistic activity (Table 8). Furthermore, crab shell powder 1.25% was used in
powder formulation containing mixture of and , because its effect is
not significant to chitin and its price is far cheaper than pure chitin

The research result was in line with previous researches in other yeasts.
Antagonistic activity of yeast (various species or isolates) can be enhanced by addition
of calcium chloride (Tian . 2002; Abadias . 2003), pure chitin (Vivekananthan

. 2004; Yu . 2007). Calcium chloride could increase the biocontrol efficacy of
tested yeast due to its ability to enhance plant resistance against plant diseases (Biggs

. 1997; Droby . 2003). The same mechanism is similar for chitin (Yu . 2007).
This research resulted in a new finding that crab shell powder, a chitin-containing
material, provides the same level of enhancement compared to pure chitin. The
mechanism how crabshell increase antagonistic activity of is not exactly
known, probably it involves induction of plant resistance. Harti (2010) reported
induced resistance of banana against fusarial wilt diseases after treated with crab shell
powder. Aside from chitin, crab shell contains protein, calcium, phosphate, and other
elements such as iron, manganese and zinc (Multazam 2002), therefore effect of
composing elements of crab shell powder on antagonistic activity could not be
ignored. This is an advantage since using crab shell powder is cheaper than pure chitin.
The use of crab shell powder does not need chemicals for processing, so it is cost-
effeective.

ne strategy to improve storability and bio-performance of formulated microbes
is by providing additives (Burges & Jones 1998; Fravel 2005; Wiyono 2008). Some
additives of antagonistic yeasts are metal ion (calcium), sugar (trehalose), biopolymer
(chitin) and calcium (Abadias . 2003). Addition of crab shell 1.25% into talc-based

C. terreus C. albidus

et al et al
et al et al

et
al et al et al

C. terreus

et al.

et al

Effect of crab shell powder as additives in talc-based powder formulation of
and

C.
terreus C. albidus

Table 9. Survival of in talc formulation and supplemented with additivesC. terreus

Yeast density (log cfu/g) in i-th month

0 1 2 3
Talc 8.7 a 6.15 a 6.54 a 5.02 a
Talc + Chitin 8.19 a 7.89 b 6.75 a 5.02 a
Talc +crab shell powder 8.73 a 7.00 ab 6.07 a 6.07 b

Numbers followed by same symbol are not significantly different at significant level of 0.05 with DMRT test

Table 10. Survival of in talc formulation and supplemented with additivesC. albidus

Yeast density (log cfu/g) in i-th month

0 1 2 3
Talc 8.87 a 6.06 a 6.50 a 5.26 a
Talc + Chitin 9.31 a 7.56 b 6.48 a 5.14 a
Talc + Crab shell powder 9.56 a 7.30 b 6.22 a 6.06 b

Numbers followed by same symbol are not significantly different at p<0.05 with DRMT test

formulation increased survival of and (Tables 9 and 10). Further
studies on the exploration and optimization of various additives are needed to prolong
survival rate of formulated yeasts

is compatible to therefore it can be used in a mixture
formulation. Best carrier materials in powder formulation of was talc, able to
maintain yeast survival for four months of storage without contamination of other
microorganisms. Talc powder provided best survival of , with survival more
than 5 months of storage. Crab shell at concentration of 1.25% can be used as additive
in powder formulation containing a mixture of and based on its
enhancement of antagonistic activity on and survival of both antagonistic
yeasts

he authors would like to acknowledge SEAMEO BIOTROP for providing
financial support through DIPA 2009. Moreover, the authors express their gratitude
to people involved in this research, Ms Ratih Munawaroh and Mr. Dadang
Surahman at the Laboratory of Plant Mycology Dept of Plant Protection, Faculty of
Agriculture, Bogor Agricultural University

C. terreus C. albidus

. terreus C. albidus,
C. terreus

C. albidus

C. terreus C. albidus,
C, terreus

CONCLUSIONS

ACKNOWLEDGMENTS

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