Dry flowable formulations of antagonistic Bacillus subtilis strain T429 by spray drying to control rice blast disease

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: DOI: Reference:

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

To appear in:

Biological Control

Received Date: Accepted Date:

25 December 2014 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 AEO5, 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 E-mail addresses: [email protected] (Y. Liu)

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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

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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.

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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.

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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

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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 suspensibility 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

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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

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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:

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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,

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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.

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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 .

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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.

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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

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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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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.

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Viability 109 cfu ml-1

5 4 3 2 1 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

Log CFU / g

8 6 4 2 0 30d

90d

180d

Storage time (d)

Fig. 2

23

360d

720d

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.

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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

26

Adhesive

Viability

Wetting time

109 cfu/g

No.

NNO

AEO-5

(NH4)2SO4

CMC-Na

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

s

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 cfu mL−1

T429 dry flowable inhibitory zone diameter cm

T429 fermentation broth

Inhibitory rate %

Inhibitory zone diameter cm

Inhibitory rate %

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