Accepted Manuscript

Dry flowable formulations of antagonistic Bacillus subtilis strain T429 by spray

drying to control rice blast disease

Xiangkun Meng, Junjie Yu, Mina Yu, Xiaole Yin, Yongfeng Liu

PII: S1049-9644(15)00032-8
DOI: http://dx.doi.org/10.1016/j.biocontrol.2015.03.004
Reference: YBCON 3235

To appear in: Biological Control

Received Date: 25 December 2014

Accepted Date: 6 March 2015

Please cite this article as: Meng, X., Yu, J., Yu, M., Yin, X., Liu, Y., Dry flowable formulations of antagonistic
Bacillus subtilis strain T429 by spray drying to control rice blast disease, Biological Control (2015), doi: http://
dx.doi.org/10.1016/j.biocontrol.2015.03.004

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Dry flowable formulations of antagonistic Bacillus subtilis
strain T429 by spray drying to control rice blast disease
Xiangkun Meng, Junjie Yu, Mina Yu, Xiaole Yin, Yongfeng Liu *

Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

ABSTRACT

Dry flowable formulations of Bacillus subtilis strain T429 with fungicidal act

ivity against the rice blast fungus Magnaporthe grisea were synthesized by s

pray drying. Inert ingredients including wetting agents, dispersants, disintegran

ts, and adhesives that show good biocompatibility with Bacillus subtilis T429

were obtained. The formulations were optimized by a four-factor and three-l

evel orthogonal experiment. The optimal contents of the wetting agent AEO-

5, dispersant NNO, disintegrant (NH4)2SO4, and adhesive CMC-Na were 1%,

9%, 5%, and 1% respectively, the filler kaolinite supplemented to 100%. T

he mixture was suspended in the fermentation broth of T429 at a ratio of 2

0% (m/v). After being ground in a ball mill for 3 h, the suspension was spr

ay-dried, and the dry flowable formulations were obtained. The formulation

showed good physical characteristics, such as high dispersibility and viability.

After 12 months of storage at room temperature, it revealed long shelf life

and high viability. Field tests in rice crops illustrated that dry flowable form

ulations at 50 and 75 g/667 m2 concentrations were as effective as a commer

cial fungicide in controlling rice blast, control efficiency

* Corresponding authors. Fax:+8625 84391002 1

E-mail addresses: liuyf@jaas.ac.cn (Y. Liu)
up to 77.6% and 78.5%, respectively. No significant differences in disease c

ontrol efficiency were observed between the formulations and the chemical p

esticide tricyclazole (79.5%). Overall, a new shelf-stable and effective dry flo

wable formulation of the biocontrol agent B. subtilis T429 was obtained by s

pray drying to control rice blast.

Keywords: Bacillus subtilis, fungicide, dry flowable, rice blast, spray drying

1. Introduction

The rice blast caused by fungus Magnaporthe grisea is a severe production

constraint worldwide (Rossman and Howard, 1990; Talbot, 2003; Ralph et al., 2005).

Infections occur when the fungal spores attach themselves to rice leaves, or the

hyphae colonize the root surface (Sylvain Marcel et al.,2010). Strategies to control rice

blast include using chemical fungicides, planting resistant rice varieties, and applying

biological control agents (Hongying Shan et al., 2013; Bohnert et al., 2004; Yuanyuan NIE et

al.,2014). Chemical applications are still the main method in controlling rice blast in

several countries; however, chemicals are harmful to the environment and can result

in fungicide resistance among pathogen populations (Todorova and Kozhuharova, 2010).

With the economic growth and increased living standards, the desire for food safety is

becoming increasingly stronger worldwide. These facts have stimulated researches on

environmental-friendly strategies to control rice blast, such as applying biological

control agents and discovering new pesticide formulations. Biological control of rice

blast is a valuable alternative to the use of chemical pesticides, and many bacterial

2
formulations have been developed to combat rice blast in developing countries

(Prabavathy et al., 2006; Krishnamurthy and Gnanamanickam, 1998; Armando et al.,2007;

Wiwattanapatapee et al.,2013) . Bacillus subtilis can produce a broad array of antifungal

lipopeptides, making this species a biological control agent (Ongena and Jacques, 2008).

Most B. subtilis formulations currently available are aqueous solutions or wettable

powders. Although they have exhibited promising biological control activity, these

formulations are disadvantaged by their short shelf life, lower stability and difficulty

in transport. It remains a major challenge to successfully develop effective

formulations and scale-up productions of the organisms (Jae et al.,2006).

Dry flowable formulations contain inert ingredients, which prolong the shelf life

and increase the efficacy of the products. On the sixth international conference of

chemical pesticides, it was reported as a new efficient, environmental-friendly

pesticide formulation. It has been considered as an important development direction

of pesticide formulations. Spray drying has been commonly used in pharmaceutical

industries, and dry flowable formulations could be synthesized by spray drying

because of its lower cost and higher energy efficiency (John M surgant, 1990;

Santivarangkna et al., 2007; Tamez-Guerra et al., 1999; Walters et al.,2014).

The endospores produced by B. subtilis are resistant to high temperatures, it

allows using spray drying as a possible avenue to prepare dry flowable formulations

of the biocontrol agent (Iryna et al., 2008; V. Ya´nez-Mendiza´ bal et al., 2012; Chung et al.,

2007). B.subtilis T429 is a microorganism whose fungicidic action has been reported.

3
It was isolated from rhizosphere soils of China as a good candidate for the biological

control of rice blast (ZHANG et al.,2011) . We previously described the optimization of

spray drying conditions of B.subtilis T429, which was shown to be a high viability

during spray drying process (Meng et al., 2014). The present study aims to produce a

dry flowable formulation of B. subtilis strain T429 , and to investigated the effects of

the formulations on the control of rice blast in petri dish and field conditions.

2. Materials and methods

2.1. Antagonistic bacterial strain and pathogens

B. subtilis strain T429 was originally isolated from the rhizosphere soil of rice

field in Nanjing, Jiangsu, China.This strain is highly fungistatic to rice fungus

diseases (Zhang et al., 2011). The pathogen Magnaporthe grisea was collected from

infected rice plants and stored in our laboratory. Potato dextrose agar medium (200 g

of potato, 20 g of dextrose, 12 g of agar, and 1000 mL of distilled water) was used as

the standard growth and storage medium for M. grisea.

2.2. Inert ingredients

Inert ingredients including dispersants, wetting agents, disintegrants, adhesives,

and the carrier kaolinite used in this study were commercial products purchased from

Suke Agrochemical of Jiangsu Province, Co., Ltd., China. The ingredients were

designated as follows: wetting agents, A1–A5; dispersants, B1–B6; adhesives, C1–C4;

and disintegrants, D1–D6. The ingredients were added to fermentation broth at the

highest recommended concentration to determine biocompatibility.

4
2.3. Optimized fermentation conditions for the antagonistic B.subtilis T429

Inexpensive, locally available raw materials such as corn starch and soybean dregs

were used for fermentation. The activated T429 was maintained on the YPG medium

(5 g L−1 yeast extract, 5 g L−1 peptone, 5 g L−1 glucose, pH 7.0) for 16 h to 18 h to

obtain the seed culture, and it was inoculated into the optimized medium: 30 g L−1

corn starch, 10 g L−1 soybean dregs, 5 g L−1 yeast extract, and 3 g L−1 K2HPO4. The

optimal fermentation conditions were as follows: initial pH, 6.8; inoculation volume,

1%; filling volume, 330 mL L−1; fermentation temperature, 30 °C; rotation speed,

160 r min−1; and fermentation cycle, 96 h. The viable counts of the fermentation

reached 3.6×109 cfu mL−1, and the spore forming rate was 95%, as examined under a

microscope.

2.4. Inert ingredients screening

Twenty-one inert ingredients, including dispersants, wetting agents, disintegrants,

and adhesives, were separately added to the fermentation broth. The sample without

any adjuvants was used as a control. All treatments were reserved at 54 °C for 2

weeks, and the viable spore counts in each suspension were detected by using the

plate count method described above.

2.5. Orthogonal optimization of formulations

Optimum formulations were selected by four-factor and three-level orthogonal

experiments (Table 1). For the orthogonal design, the ratios of dispersants, wetting

5
agents, disintegrants, and adhesives were determined on the basis of the viability and

wetting time.

2.6. Wet grinding and spray drying of T429 formulations

The formulations were prepared with four optimized inert ingredients and then

suspended in the fermentation broth of T429. The carrier kaolinite was added to the

suspension to make the solid concentration reach 200 g L−1. Finally, the suspension

was ground in a ball mill for 6 h to ensure homogeneity and dried by a laboratory

spray dryer (YC-1000 Spray Dryer, Yacheng, Shanghai, China). Spray drying

conditions were selected based on previous tests with B.subtilis as follows (MENG et

al., 2014).: inlet air temperature, 115 °C; feed flow rate, 720 mL h−1; atomization

pressure, 0.1 MPa; and hot air flow, 30 M 3 h−1. The dry flowable products was

collected from the cyclone collector and stored in a jar at room temperature.

2.7. Physical characteristics and storage stability of T429 formulations

Physical characteristics such as wetting time, pH, dispersibility, and suspen-

sibility were detected in accordance with the standards of the Collaborative

International Pesticides Analytical Council (CIPAC).

2.7.1. Storage stability

Biological activity either remains constant or decreases with storage time. To

study stability, 10 g of the spray-dried formulations was stored in 50 mL screw-cap

conical polypropylene tubes. The containers were sealed with Parafilm and then

6
stored in an airtight container filled with silica gel to avoid sample humidification.

The samples were stored 20 ± 5 °C, and each treatment was repeated in triplicates.

2.7.2. Wettability

A 100 mL aliquot of standard hard water (CIPAC, MT18; CIPAC 1970) was

transferred into a 250 mL beaker (Lisansky et al. 1993). A representative sample of

the powder (0.1 g) was added at once by dropping it on the water via a glass funnel

held in a ring stand. The bottom of the funnel was 10 cm from the surface of the water.

The stopwatch was started, and the time taken (to the nearest second) for it to become

completely wetted was recorded. The experiment was repeated three times, and the

result was averaged.

2.7.3. Moisture Content

The moisture content of all samples was determined by drying in a moisture

analyzer (Sartorious MA 30, Goettingen, Germany) to a constant weight at 105 °C.

2.7.4. Bacterial viability

Viable bacteria (c.f.u) of the culture broth and spray-dried formulations were

determined by using the plate count method. The formulation (1 g) was dissolved in

99 mL of sterile distilled water, gently mixed, and then serially diluted to 10−9.

A 0.33 mL aliquot from the 10−7, 10−8, and 10−9 dilutions was plated on each of three

YPGA plates and then cultured at room temperature for 24 h, after which the c.f.u was

counted.

2.8. Field experiments

7
 The field experiments were carried out in two separate rice fields in Huaian and

Nanjing area, Jiangsu Province. Rice varieties Huaidao No 5 and Nangeng 46, which

generally planted in the two areas, were used in the field experiments. The two

experimental fields were restricted and each field was artificially infested with M.

grisea before applying any formulations . In each case, the field was divided into 21

plots, each plot occupied 30 m2. A completely randomized design was used with three

replicates of seven treatments The treatments were as follows: (1) T429 dry flowable,

25 g/667 m2; (2) T429 dry flowable, 50 g/667 m2; (3) T429 dry flowable, 75 g/667 m2;

(4) 75% tricyclazole, 25 g/667 m2; (5) 6% kasugamycin, 25 g/667 m2; and (6) control.

2.9. Statistical analysis

The rice blast scores were recorded according to the Standard Evaluation System

(1-9 scale) of Intermational Rice Research Institute (IRRI, 2002; Yuanyuan NIE et

al.,2014 ). The rating scale used to measure rice panicle blast disease severity as

follows: Scale 0, No visible lesion observed or lesions on only a few pedicels; 1,

lesions on several pedicels or secondary branches; 3, lesions on a few primary

branches or the middle part of panicle axis; 5, lesion partially around the base or the

uppermost internode or the lower part of panicle axis near the base; 7, lesion

completely around panicle base or uppermost internode or panicle axis near the base

with more than 30% of filled grains; 9, lesion completely around panicle base or

uppermost internode or the panicle axis near the base with less than 30% of filled

grains. Disease index and control efficacy were calculated as follows:

8
Disease index =Ʃ (rice blast rating×number of panicles at that rating)×100/(total

number of rice panicles investgated×9)

Control efficacy= (Disease index of the control - Disease index of the treatment) /

Disease index of the control ×100%.

ANOVA was performed by SPSS version 19.0 computer software package to

test the significance. All datas were compared with Duncan’s Multiple Range Test.

Statistical significance was considered at P < 0.05.

3. Results
3.1. Inert ingredients screening

The effect of different inert ingredients on the survival of the B.subtilis T429 were

presented in Fig.1. After stored at 54 °C for 2 weeks, the number of viable spores

declined rapidly. Most of the inert ingredients inhibit the viability of B. subtilis. A5

and B6 exhibited detrimental effects on the spores, no viable

bacterium could be detected after 2 weeks of storage at 54 °C.

Meanwhile, A2, B3, C3, D2, and D4 did not significantly influence

spore viability compared to 54-CK,viable bacterium remained 2 to 2.5 ×109cfu/ml.

Thus they could be employed as components of the formulations. Comparison

between RT-CK and 54-CK showed that the spore viability of the

latter sample was significantly lower than that of the former sample.

This result confirmed that long-term high temperature is still an

important limiting factor for the storage of living spores.

9
3.2. Orthogonal optimization of formulation

The effects of different factors on viability were compared by a four-factor and

three-level orthogonal test . The addition of the dispersant NNO (A2), wetting agent

AEO-5 (B3), and adhesive CMC-Na (C3) significantly increased viability（Table 2）.

Among these ingredients, C3 exerted the greatest effect. The viability values were

2.837, 3.037, and 2.66 (×109 cfu mL−1) when the ratios of the disintegrant (NH4)2SO4

(D2) were 3, 5, and 7, respectively. These results indicate that the suitable ratios of the

wetting agent AEO-5 (A2), dispersant NNO (B3), adhesive CMC-Na (C3), and

disintegrant (NH4)2SO4 (D2) were 9%, 3%, 5%, and 1%, respectively.

For the wetting time, comparison between the different factors illustrated that

excellent wettability (< 30 s) was obtained. With increasing wetting agents, less time

was needed for the products to become completely wetted. Yields of wetting time

were 20.333, 20.467, and 19.733 when the ratios of AEO-5 were 1%, 2%, and 3%.

Viability is an important parameter of biological pesticides. To increase the

wettability of the products, the formulation should contain the following components:

1.5% wetting agent AEO-5, 9% dispersant NNO, 1% adhesive CMC-Na, and 5%

disintegrant (NH4)2SO4.

3.3. Physical characteristics of dry flowable formulations

Dry flowable formulations of B. subtilis T429 were successfully developed by

orthogonal optimization. The formulations containing various adjuvants of NNO,

10
AEO, CMC-Na, and (NH4)2SO4 illustrated better physical properties (Table 3) in

accordance with the standards of CIPAC.

3.4. Storage stability

Shelf life is an important index of biological pesticides. The products was

collected in screw-cap conical polypropylene tubes sealed with Parafilm and stored at

room temperature for 2 years As shown in Fig. 2, the vial spore density was

1.145×109 cfu g−1 and the viability was 90.16% after 720 d. The decline in viability

was not significant.

3.5. Inhibition to M. grisea mycelial growth

Compared with the fermentation broth, the aqueous solution of the formulations

had almost equal ability to inhibit the mycelial growth of M. grisea at the same

concentration (Table 4). Between the effective concentration 107 to 108 cfu mL−1

during the field use, the inhibitory rates of the T429 dry flowable and fermentation

broth were 63.4% to 64% and 64.2% to 64.7%, respectively.

3.6. Field experiments

In the field experiments, the application of dry flowable formulations containing B.

subtilis by spraying significantly reduced the development of rice blast (Table 5).

In Nanjing and Huaian, the disease index of rice blast with T429 at 75 g/667 m2 were

0.85 and 0.19, and the control efficiencies were 77.32% and 77.6% respectively,

which were higher than the control efficiencies of 25 g/667 m2 and 50 g/667 m2.

11
Conversely, the treatments with T429 dry flowable at 75 g/667 m2 were as effective as

the 75% tricyclazole and 6% kasugamycin at 25 g/667 m2 in both areas.

4. Discussion

As an alternative to chemical pesticides, B. subtilis is considered as an important

biocontrol agent. It has the potential to produce a number of antibiotics and has been

demonstrated to have a strong inhibiting capacity to plant pathogenic fungi and virus

(Chuping Luo et al.,2015; Xiaojia Hu et al.,2014). Our laboratory has developed B.

subtilis to control the fungal disease in rice for many years. We have previously

produced commercial liquid formulations to control sheath blight and false smut in

rice for the Chinese market (Su-wen et al., 2004). Although liquid formulations of

bacteria antagonists have been proven efficient against rice disease, the applications

of these formulations are limited by their short shelf life and difficulty in transport.

Recently, spray drying has been introduced to develop new formulations of

antagonists, such as Bacillus thuringiensis, B. subtilis, and even virus (Arthurs et al.,

2006; Kun-Nan et al., 2013; Ya´nez- Mendiza´bal, 2008; Vin et al., 2011).

To expand the application of B. subtilis, spray drying was applied to

produce products that meet the standard of commercialization and to

provide a satisfactory control efficacy against rice blast. Here, this work

investigated the biological control activity of the improved dry flowable formulations

of B. subtilis T429 to control rice blast. Spray drying is expected to be efficient for

producing antimicrobial products .

12
 To develop a successful formulation protocol for biological control agents,

selection of appropriate carrier materials and adjuvants is important. Because they

are often added to biopesticides to improve the efficiency, suspensibility

dispersibility, and adhesive ability on plant leaves (Zhenhua et al, 2011)., Considering

that live bacteria are the basis of biopesticides, we must evaluated the potential

adverse effects of adjuvants on viability. In our study, 21 types of adjuvants were

added to the fermentation broth to detect the biocompatibility with B. subtilis. The

results demonstrated that different types of adjuvants have varying effects on spore

viability. Considering spore viability, we selected the wetting agent AEO-5,

dispersant NNO, adhesive CMC-Na, and disintegrant (NH4)2SO4 as the appropriate

components. To establish dry flowable formulations, the components and their levels

should be selected. The orthogonal test is widely used in optimizing pesticide

formulations (Liu et al., 2009). A four-factor and three-level orthogonal test was

carried out on the basis of the viability and wetting time.

Ball mill grinding is widely used by pharmaceutical or pesticides

industries to homogenize the ingredients and reduce the particle size

(Naoya et al., 2011). Our research illustrated that smaller particle

sizes and better suspensibility can be obtained by ball milling, whereas

the viability of B. subtilis T429 spores was not reduced during ball

milling.

13
 Compared with other formulations, the current dry flowable formulations were

preferred over wettable powders and liquid formulations.Because the former

contained large quantities of active adjuvants, it dispersed homogeneously in

water immediately without dusts. The dissolved suspension was easily

attached to the plant leaves after sprayed because of the adhesive ingredients.

Potent formulations with high ingredient contents were less costly to ship and

transport to distant areas than liquid formulations.

In our room experiments, the dry flowable formulations was highly effective in the

control of rice blast, as compared to the fermentation broth. Field experiments were

conducted in two restricted rice fields, in order to make the rice blast occurred

uniformly, the rice was artificially infested with M. grisea before applying any

treatment. The results illustrated that many environmental factors in the field

experiments might have contributed to the inconsistency in disease control. There

were differences in disease index and control efficiency between Nanjing and Huaian,

Nevertheless, the rice plants applied with dry flowable formulations of B. subtilis

T429 illustrated considerably higher control efficiency than the control group, in

which the severity of disease was the greatest. The suppression of rice blast by

applying the dry flowable formulation was as effective as that by applying the

chemical fungicide tricyclazole and the agricultural antibiotic kasugamycin.

In our study, we employed a cost-effective medium for fermentation. Locally

available raw materials including corn starch and soybean dregs were used for the

14
large scale production of B.subtilis T429. Consistent with previous reports, the present

study indicates that the dry flowable formulation of T429 is a promising alternative to

control rice blast. It is expected to be efficient and helpful in the application of

antagonistic bacteria as fungicides.

Acknowledgment

This study was supported by Key Technology Support Program of Jiangsu

province (BE2014386) and “948” program from Ministry of Agriculture of

P.R.C.(2011-G4).

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Zahra Shokri, Mohammad Reza Fazeli., Mehdi Ardjmand, Seyyed Mohammad Mousavi, et al.

Factors affecting viability of Bifidobacterium bifidum during spray drying. DARU Journal of

Pharmaceutical Sciences.

19
ZHANG Fen, 2011. Screening and identification of antagonistic bacteria against Pyricularia

grisea Jiang su J. of Ag r. Sci 27( 3 ),505~ 509.

Zhenhua Liu , Honggang Wei , Yuanguang Li , Shulan Li , Lin Zhang & Houlong Chen, 2011.

Effects of milling and surfactants on suspensibility and spore viability in Paenibacillus

polymyxa powder formulations. Biocontrol Science and Technology 21, 1103-1116.

20
Figure captions

Fig. 1. Effect of inert ingredients on the survival of B. subtilis T429 spores during 2 weeks of

storage at 54 °C. A1–A5, dispersants; B1–B6, wetting agents; C1–C4, adhesives; D1–D6,

disintegrants. RT-CK and 54-CK are samples without any adjuvants stored at room temperature

and 54 °C, respectively. Error bars represent standard deviations.

Fig. 2. Effect of storage time on the survival of B. subtilis T429 spores during reserved at room

temperature for 2 years. Error bars represent standard deviations.

21
 6

Viability 109 cfu ml-1 5

0
RT- 54- A1 A2 A3 A4 A5 B1 B2 B3 B4 B5 B6 C1 C2 C3 C4 D1 D2 D3 D4 D5 D6
CK CK
Inert ingredient No.

Fig. 1

22
 10

8
Log CFU / g

0
30d 90d 180d 360d 720d
Storage time (d)

Fig. 2

23
Table captions

Table 1 Orthogonal design matrix with four inert ingredients

Table 2 K-values and ranges of four inert ingredients for the optimization of the formulations

Table 3 Physical properties of dry flowable formulations

Table 4 Inhibitory rate of viable cells (±SD) (cfu mL−1) on the hyphal growth of M. grisea and

control efficacy

Table 5 Control efficiency of B. subtilis T429 dry flowable in suppressing the rice blast disease

in the field.

24
Table 1

Level Dispersant Wetting agent Disintegrant Adhesive

1 3 0.5 3 0.3

2 6 1 5 0.5

3 9 2 7 7

25
Table 2

Treatment Dispersant Wetting agent Disintegrant Adhesive Viability Wetting time

26
 No. NNO AEO-5 (NH4)2SO4 CMC-Na 109 cfu/g s

1 3 0.5 3 0.3 2.31 20.9

2 3 1 5 0.5 2.75 17.9

3 3 1.5 7 1 3.18 18.7

4 6 0.5 5 1 3.30 21.7

5 6 1 7 0.3 2.43 19.2

6 6 1.5 3 0.5 2.74 19.2

7 9 0.5 7 0.5 2.37 18.4

8 9 1 3 1 3.46 24.3

9 9 1.5 5 0.3 3.06 21.3

K1 2.747 2.660 2.837 2.600

K2 2.823 2.880 3.037 2.620

K3 2.963 2.993 2.660 3.313

R 0.216 0.333 0.377 0.713

K1 19.167 20.333 21.467 20.467

K2 20.033 20.467 20.300 18.500

K3 21.333 19.733 18.767 21.567

R 2.166 0.734 2.700 3.067

K1: total of each factor in its first level, K2: total of each factor in its second level, K3: total of

each factor in its third level, and R: range of each factor in its each level.

Table 3

27
Properties Description

Spore viability (cfu g−1) ≥ (1.0±0.2) × 1010

Wetting time (s) 25 s

pH (6.8±0.2)

Suspensibility (%) 85

Moisture Content (%) 4.5

28
Table 4

Treatment T429 dry flowable T429 fermentation broth

cfu mL−1 inhibitory zone diameter Inhibitory rate Inhibitory zone diameter Inhibitory rate

cm % cm %

109 3.15a 70 3.14a 69.8

108 2.88ab 64 2.91ab 64.7

29
 107 2.86ab 63.6 2.89ab 64.2

106 2.73abc 60.7 2.81ab 62.4

* Means followed by the same letter show no significant difference by Duncan’s multiple range

test at P < 0.05.

30
Table 5

Treatment Nanjing Huaian

Disease index Control efficiency (%) Disease index Control efficiency (%)

1 1.27 65.90c 0.33 62.2c

2 1.12 69.87c 0.32 63.1c

3 0.85 77.32ab 0.19 77.6ab

4 0.67 82.04a 0.17 79.5a

5 1.19 68.22c 0.18 79.1a

6 3.73 - 0.86 -

* Means followed by the same letter show no significant difference by Duncan’s multiple range

test at P < 0.05

31
Antagonistic B.subtilis strain T429 with fungicidal activity was spray dried

to control rice blast under field conditions.

32
1 Dry flowable formulations of Bacillus subtilis strain T429 were synthesized by

spray drying.

2 Wetting agents, dispersants, disintegrants, and adhesives that show good

biocompatibility with B. subtilis T429 were obtained.

3 Dry flowable formulations were shelf-stable and effective to controll rice blast.

33