Bacillus subtilis

2559
 DEVELOPMENT OF FORMULATION AND

APPLICATION OF Bacillus subtilis FOR CONTROLLING

SOFT ROT DISEASE OF CHINESE MUSTARD

Jitmanus Nikaji

A Thesis Submitted in Partial Fulfillment of the Requirements for the

Degree of Master of Science Program in Crop Science

Suranaree University of Technology

Academic Year 2016

 Bacillus subtilis

(
 : Bacillus subtilis
(DEVELOPMENT OF FORMULATION
AND APPLICATION OF Bacillus subtilis FOR CONTROLLING SOFT ROT
DISEASE OF CHINESE MUSTARD) :
, 121 .

Bacillus subtilis
CaSUT007 CaSUT007 (Brassica juncea)
3 molasses diamonium
phosphate yeast extract (MDY) 1.30±0.16x109cfu.ml-1
48
spray dry
3 MDY
6 24
8.2×107 cfu.ml-1
SJN007 0.5 1
1.7×108 cfu.ml-1
5 (K2S2O5)
250 ppm 6 24
1.0±0.14×101 cfu.ml-1
starter)
1:10
1.47±0.86×108 cfu.ml-1
Erwinia carotovara pv. carotovora
MAX018 9
SJN007 1:50 3 10, 20
30
92 Bacillus
69 27

FT-IR spectroscopy
 lipid triglycerides (1739 cm-1) lipids (1460 cm-1)
amino acid fatty acids (1406 cm-1) ß-sheet protein (1631
cm-1) amide I (1642 cm-1) amide II (1560 cm-1
pectin lignin SJN007
B. subtilis CaSUT007

___________________________
2559 _____________________
 JIDMANUS NIKAJI : DEVELOPMENT OF FORMULATION AND

APPLICATION OF Bacillus subtilis FOR CONTROLLING SOFT ROT

DISEASE OF CHINESE MUSTARD. THESIS ADVISOR : ASST. PROF.

NATTHIYA BUENSANTEAI, Ph.D., 121 PP.

CHINESE MUSTARD/Bacillus subtilis/SOFT ROT/Erwinia carotovora/

FORMULATION/APPLICATION/FTIR SPECTROSCOPY

The aim of this study was to develop formulation and application of Bacillus

subtilis strain CaSUT007 to control soft rot disease in Chinese mustard (Brassica

juncea). The strain CaSUT007 was cultured in 3 different media. It was found that

molasses supplemented with diamonium phosphate and yeast extract (MDY) gave the

highest concentration of bacteria at 1.30±0.16x109 cfu.ml-1 after 48 hr. The CaSUT007

was subsequently formulated into a spray dry starter formulation (JN007 bioproduct)

under a suitable air temperature. Then, the JN007 starter was cultured in 3 different

multiplication media, and the bacterial growth was monitored. It was found that the

MDY medium gave the highest concentration of CaSUT007 cells at 8.2×107 cfu.ml-1

after 24 h with twice shaking at 6 h. A suitable ratio of the JN007 starter to the

multiplication medium was observed at 0.5 g per 1 liter, yielding the cell

concentration of 1.7×108 cfu.ml-1 after 24 h. Five different decontamination methods

were tested to reduce other microbial populations in the multiplication medium. It was

found that 250 ppm of potassium metabisulfite could reduce the contamination

significantly, having only 1.0±0.14×101 cfu.ml-1 of other contaminants compared to

none when the standard autoclave was used. The ratio of multiplied culture and

multiplication media was observed, and it was found that the ratio 1:10 gave maximum
bacterial cell at the concentration of 1.47±0.86×108 cfu.ml-1. The formulation was

further tested for its induced resistance ability against soft rot bacterial pathogen,

Erwinia carotovara pv. carotovora (ECC) in Chinese mustard, MAX018 cultivar

under a greenhouse condition. The result revealed that seed soaking combined with

root dipping and foliar spray at 10, 20 and 30 days after planting with the

multiplication culture of JN007 at the ratio 1:50 showed the significantly highest

percentage of disease reduction at 92% compared to those of the chemical and

commercial Bacillus, which were only 69 and 27%, respectively. Biochemical

component changes in the treated Chinese mustard leaves were analyzed at 7 days

after the ECC challenged inoculation by FT-IR spectroscopy. The result revealed

higher amount of lipid and triglycerides (1739 cm-1), lipids (1460 cm-1), amino acid and

fatty acids (1406 cm-1), ß-sheet protein (1631 cm-1), protein amide I (1642 cm-1), and

amide II (1560 cm-1 The biochemical changes were suggested to associate with pectin

and lignin accumulation, making the induced Chinese mustard more resistant to soft

rot disease. Therefore, the starter formulation SJN007 of B. subtilis strain CaSUT007

can be effectively used to reduce soft rot disease.

School of Crop Production Technology Student Signature__________________

Academic Year 2016 __________________

1. 1
1.1 1
1.2 2
1.3 2
1.4 2
1.5 3
2. 4
2.1 4
2.2 Bacillus sp. 5
2.2.1 (taxonomy) Bacillus sp. 6
2.2.2 Bacillus sp. 6
2.2.3 Bacillus 6
2.3 Bacillus 8
2.3.1 (competition) 8
2.3.2 (antibiosis) 9
2.3.3 (parasitism) 11
2.3.4 (induced resistance) 12
2.4 induced resistance: IR) 13
 2.4.1 (structural defenses mechanism) 14
2.4.2 biochemical defenses) 15
2.4.2.1
pre-existing biochemical defense) 15
2.4.2.2
induced biochemical defense) 15
2.5 (IR inducers/ elicitor/activators) 15
2.5.1 (PGPR)
16
2.5.2
synthetic resistance inducer) 19
2.5.2.1 Benzo 19
2.5.2.2 salicylic acid, SA) 20
2.5.2.3 thiamine) 20
2.5.2.4 jasmonic acid, JA) 20
2.5.3
natural resistance inducer) 21
2.6 22
2.6.1 22
2.6.2
infrared (IR) spectroscopy 23
2.7 B. subtilis 28
2.7.1 28
2.7.2 29
2.7.3 30
2.7.4 30
2.8 B. subtilis 31
 2.8.1 liquid formulate) 31
2.8.2 dry formulate) 32
2.9 33
2.10 spray dry) 34
2.10.1
37
2.10.1.1 38
2.9.10.2 38
2.11 B. subtilis 39
2.11.1 carrier) 39
2.11.2 binder) 39
2.11.3 protectant) 40
2.12 B. subtilis 41
2.13 42
2.13.1 physical method) 42
2.13.1.1 dry heat) 42
2.13.1.2 moist heat) 43
2.13.1.3 radiation) 44
2.13.1.4 filtration) 44
2.13.2 chemical method) 44
3. 47
3.1 B. subtilis 47
3.1.1 B. subtilis 47
3.1.2 B. subtilis
spray dry) 47
3.1.3 B. subtilis
48
 3.2 B. subtilis 48
3.2.1
48
3.2.2
starter)
mortification media) 49
3.2.3
B. subtilis CaSUT007
49
3.2.4
50
3.2.5
50
3.2.6 51
3.2.7
Infrared (IR) Spectrometer 52
3.3 53
4. 54
4.1 B. subtilis 54
4.1.1 B. subtilis 54
4.1.2 B. subtilis
spray dry) 55
4.1.3 B. subtilis
57
4.2 B. subtilis 59
4.2.1
59
 4.2.2
starter)
mortification media) 62
4.2.3
B. subtilis CaSUT007
66
4.2.4
67
4.2.5
68
4.2.6 70
4.2.7
Infrared (IR) Spectrometer 73
5. 76
96
116
121
1
19
2 27
3 Bacillus
34
4 Bacillus sp. 41
5 PMS 46
4.1 B. subtilis 4 54
4.2 B. subtilis
3 57
4.3 B subtilis CaSUT007 spray dry
6 58
4.4 B. subtilis
CaSUT007 6 59
4.5
B. subtilis CaSUT007 61
4.6
B. subtilis CaSUT007 61
4.7
B. subtilis CaSUT007 62
4.8 63
4.9 starter)
mortification media) 65
4.10
B. subtilis 69
4.11
B. subtilis 71
5.1 Bacillus spp. 81

1 B. subtilis CaSUT007
118
2 B. subtilis
CaSUT007 119
3 nutrient glucose broth NGB)
molasses diamonium phosphate and yeast extract MDY) 120
4
B. subtilis CaSUT007 120
1 (competition) B. subtilis 9
2 (antibiosis) B. subtilis 11
3 (parasitism) B. subtilis 12
4 (induced resistance) 13
5 (structural defenses mechanism) 14
6 26
4.1 100X
B. subtilis CaSUT007 55
4.2 B. subtilis CaSUT007
2 spray dry 56
4.3 B. subtilis CaSUT007 spray dry
58
4.4 64
4.5 starter)
murtification media) 65
4.6 Molasses diamonium phosphate and yeast extract (MDY)
B. subtilis 66
4.7
67
4.8

E. carotovara pv. Carotovora 72

4.9 Second derivative
B. subtilis CaSUT007 (SJN 007)
2005-759 cm -1 74
4.10 Principal component analysis (PCA)
B. subtilis
CaSUT007 (SJN 007) 75
5.1 80
5.2 91
5.3 93
5.4
B.subtilis CaSUT007 94
5.5 B.subtilis CaSUT007 95

1 600
(Log of cell (cfu.ml-1) B. subtilis CaSUT007 118
2 B. subtilis CaSUT007
119
 1

1.1
Plant
growth promoting rhizobacteria : PGPR
Bacillus sp.

induced systemic resistance : ISR , 2552; , 2552;

Kloepper et al., 2004; Choudhary et al., 2009) B. subtilis
B. subtilis
, 2550; ,
2556; Jayaraj et al., 2006 Bacillus
B. licheniformis SP009s

84
, 2551 B. firmus
37.4-51.3 5.60-22.44
(
, 2546 2557 B. subtilis
CaSUT007 200 ppm
47.1
B. subtilis fresh culture
(powder formulation)
1)
2)
 2

(inoculum) (starter medium)

(multiplication media)

Bacillus sp.

Erwinia carotovora pv. carotovora

1.2
1. B. subtilis
(spray dry)
2. B. subtilis

1.3
1. B. subtilis

2. B. subtilis

1.4
B. subtilis
CaSUT007
Erwinia carotovora
pv. carotovora
 3

1.5
1. B. subtilis

2. B. subtilis
 2

2.1
Brassica juncea L) Tindall, 1968; Bantoc, 1970
Leaf mustard Indian mustard Chinese mustard ( ,
2544 Swatow mustard ( , 2524
3

950
2558
90
300 10-15 , 2558

Erwinia carotovora pv. carotovora (New name: Pectobacterium

carotovorum sub sp. carotovorum) ( , 2551; Hauben et al., 1999 )

, 2555; Zhao et al., 2013

50
6-24
, 2556
( 2553
Zhao et al., 2000

(Doane and Chapman, 1964)

 5

copper hydroxide 30-50 20 7-10

( , 2537 ; , 2545)

Burr et al., 1988)

2.2 Bacillus sp.

Bacillus
endospore)

-5 75 pH 2-8 NaCl
25 55
B. subtilis
bacillomycin, iturin,
mycosubtilin, bacilysin, fengymycin mycobacillin
glucanase
glucans chitinase Bacillus
subtilis siderophore
ferric iron receptor)

Bacillus sp. PGPR

( , 2552; , 2559;
Rajet et al., 2003; Domenech et al., 2006; Kumar et al., 2011)
 6

2.2.1 ( Taxonomy) Bacillus sp.

Subkingdom : Firmicutes
Division : Bacilli
Order : Bacillales
Family : Bacillaceae
Genus : Bacillus
Species : Bacillus subtilis
Binomial name : Bacillus subtilis

2.2.2 Bacillus sp.

Bacillus bacteria) rod shape) Gram
positive bacteria) Bacillaceae Clostridium Desulfotomaculum
flaggella) aerobic bacteria)
facultative anaerobe thermoduric bacteria)
spore forming bacteria) bacterial spore) Bacillus
(
, 2551; Frandberg et al., 1994; Brooks et al., 2015)
Bacillus protiolytic bacteria
microbial spoilage)
B. subtilis (
, 2551; Frandberg et al., 1994; El-Hassan and Gowen, 2006; Brooks et al., 2015)
2.2.3 Bacillus
B. subtilis

1996 Asaka and Shoda

B. subtilis RB14
iturin A surfactin 2
R. solani Brian et al., (2002)
Bacillus M04 Botritys cinerea (grey mould), Ralstonia
solanacearum (bacterial wilt) E. carotovora pv. carotovora (bacterial soft rot)
 7

extracellular methanol
212 cyclic antibiotic lipopeptides iturin
Pseudomonas fluorescens SP s B. subtilis SP s
Alternaria
Alternaria
52.6, 75.0, 28.0 79.7
82.5, 21.0 33.3 41.7
, 2549; , 2550; Prathuangwong et al., 2004; Prathuangwong
et al., 2005 B. amyloliquefaciens KPS46 P. pabuli SW01/4
Alternaria sp. 43.0, 52.0
45.0 91.0, 45.5 39.8
30.8 Prathuangwong et al., , Prathuangwong et al.,
a; Prathuangwong et al., 2006b) Cho et al. (2003)
B. subtilis KS03
Gloeosporium gloeosporioides B. subtilis
KS03 iturin A2
Swain et al. (2006) Liu et al. (2006 B. subtilis
Cicerarietinum 70 74
2549 B. amyloliquefaciens KPS46

SMV SCLV)
2551
B. amyloliquefaciens KPS46

4
KPS46 20-40

Spencer X. axonopodis pv. glycines

KPS46 defense related proteins
 8

2557
B. subtilis CaSUT007
B. subtilis CaSUT007

2559 B. subtilis TU-

Orga1
TU-Orga1 X. compestris pv. compestris (Xcc)
Alternaria sp. 5.0-5.1 80.0-83.3 Berger et al.
( B. subtilis photinia
brassica

2.3 Bacillus sp.

4
2.3.1 (competition)

biocontrol agent
biocontrol
agent
1
micronutrient
Fluorescent Pseudomonads
(site of infection)
( ,
2534) P. fluorescent siderophore
Gaeumannomyces graminis var. tritici Take-all

B. cereus UE8
 9

Phytopthora spp.
zoospore Gilbert et al., 1990

1 (competition) B. subtilis
: Bautista et al., 2014)

2.3.2 (antibiosis)

(Toxin) (antibiotic)
(antibiotic)
B. subtilis PBRS-1
B. subtilis AP-3 5 Ralstonia solani,
Colletotrichum truncatum, Sclerotinia sclerotiorum, Macrophomina phaseolina Phomopsis
sp. Bacillus iturin B.
subtilis antibiotic 60
B. subtilis

Araujo et al., 2005 B. subtilis A-13

9
(toxin)
lypopeptide Bacillus Pseudomonas

biofilm (Raaijmakers et al.,

 10

2010)
2 iturin A
surfactin surfactin iturin A

(Manjula et al., 2005)

Ting (2009) B. pumilus strain BSH-4
D. bryoniae, A. solani R. solani
(stable)
100 1 UV 10
pH 6-8

1996 Asaka and Shoda B. subtilis RB14

iturin A surfactin 2
R. solani
Sharga (1998 B. subtilis BS107

B. subtilis BS107 Nuclease),

Protiase) Lipase) E. carotovora sub sp.
atroseptica E. carotovora sub sp. carotovora B. subtilis BS107

4
1
 11

2 (antibiosis) B. subtilis
: https://lookfordiagnosis.com/mesh_info.php?term=antibiosis&lang=1

2.3.3 (parasitism) parasite)

R. solani
B. subtilis RB14-C
flutolanil B. subtilis RB14-C flutolanil
flutolanil 375
94
(Kondoh et al.,2000) (2551)
Bacillus sp. 503
Cercospora cruenta, Uromyces vignae Oidium sp. B. megaterium
HT-NK-460 B. brevis TZ-CP-342
C. cruenta, U. vignae Oidium sp. 97.22 100 B. subtilis 2
rifampicin
B. subtilis 2 35.85 65.52
 12

3 (parasitism) B. subtilis
: http://www.yourarticlelibrary.com/bacteria/useful-notes-on- parasitic-saprophytic-and-
symbiotic-bacterias/6791/

2.3.4 (induced resistance)

(elicitor)

(
, 2550)
defense mechanism
cellulose hemicelluloses
cork layers)
gums)
abscission layer Agrios, 1997) Shetty et al. (2009)
-1,3-glucan Septoria tritici
callose -1,3-glucansae B.
subtilis (induced disease resistance)
toxic metabolite
Rhizoctonia solani Sclerotinia sclerotrium ( , 2535)
(2557) Bacillus sp.
CaSUT007 4
blotter method CaSUT007 200 ppm
 13

MAX018 CaSUT007
200 ppm 5
Bargabus 2002 B. mycoides strain Bac J.
B. pumilus strain 203-6 sugar beet peroxidase, chitinase -1 3-
glucanase Bacillus
B. subtilis, B. pasteurii, B. amyloliquefacieus, B. cereus, B. pumilus, B.
mycoides B. sphaericus

Kloepper et al., 2004 B. pumilus

Cucumber mosaic virus Zehnder et al.,

4 (induced resistance)
: https://www.researchgate.net/figure/271389442_fig1_Fig-1-Scheme-of-different-
types-of-systemic-resistance-The-systemic-acquired

2.4 (induced resistance: IR)

 14

(plant defense mechanisms)

2 (structural defense
mechanism) (biochemical defense mechanism)

2.4.1 (structural defenses mechanism)

(wax)
cuticle

(
, 2554)
glycoprotein
cutinase, cellulose hemicellulase

5 (structural defensesmechanism)
: Kumar, 2013
 15

2.4.2 (biochemical defenses)

Agrios, 1997)
2.4.2.1 (pre-existing biochemical
defense)
(fungitoxic exudates)
phytoanticipins hydrolytic enzymes chitinase
glucanase Agrios, 1997
2.4.2.2 induced biochemical
defense)
(elicitor) glycoprotein, fatty acid, carbohydrates peptides

1) hypersensitive response
2)
salicylic acid (SA) HR
pathogenesis-related (PR) protein (Ryals et al., 1996; Maleck et al., 2000; Wang et al., 2006;
Vleesschauwer et al., 2008) 3) reactive oxygen species (ROS)
5 superoxide hydrogen peroxide (H2O2)
(Thordal-Christensen et al., 1997; Shetty
et al., 2009) 4) nitric oxide (NO) ROS
5) pathogenesis related proteins (PR proteins)
( , 2554; Van Loon, 1997;
Chen et al., 2000)

2.5 (IR inducers/ elicitors/ actvators)

(inducers / elicitors/ actvators) 2

 16

( ) (coat protein
, glucan chitin )
2.5.1 (plant growth
promoting rhizobacteria, PGPR)
PGPR Pseudomonas,
Azospirillum, Azotobacter, Bacillus, Burkholderia, Gluconacetobacter Serratia
PGPR
2

phytohormones auxin, cytokinin, gibberellin

(siderophores)

(antibiotic)
antifungal metabolites

(siderophores) ,
2555 Domenech (2006) B. subtilis B.
licheniformis
B. subtilis E7-17
3.12 /
1.75 / 2008
Buensanteai
extracellular proteome B amyloliquefaciens KPS46

B amyloliquefaciens KPS46 Buensanteai et al., 2009

(2551) P. fluorescens
SP007s B. licheniformis SP009s
2
 17

Kloepper et al. (1980) Weller (1983) Chickpea PGPR

B. subtilis AF1 P. fluorescent RBT13 Fusarium sp.

Serratia plymuthica R1GC4

Pythium ultimum ISR
(induced systemic resistance)
Chunquan et al. (2000)
root and crown rot Pythium aphanidermatum PGPR
P. corrugata 13
phenylalanine ammonia-lyase (PAL) (precursor)
phytoalexins
16 peroxidase (PO) polyphenol
oxidase (PPO) PGPR 2-5

P aphanidermatum
Buensanteai et al. (2008) Buensanteai et al. (2009) B.amyloliquefaciens
Indole-3-acetic acid
(IAA) 30
glucanase, peroxidase, phenylalanine ammonia lyase,
salicylic acid, jasmonic acid defense
mechanism X. axonopodis pv.
glycines
Liang et al. (2011) B. megaterium L8 PGPR
P. aphanidermatum
B. megaterium L8
83.45 31.68 28
2550 2
Bacillus sp. IN937a IN937b plant growth promoting rhizobacteria:
PGPR mixtures IN937a+b total peroxidase
tatal superoxide dismutase
 18

Buensanteai 2009
PGPR B. amyloliquefacien KPS46
X. axonopodis pv. glycines Phytohormones
jasmonate salisaylate SAR ISR
pathways Vallad and Goodman, 2004; Buensanteai et al., 2009
2557 Bacillus sp. CaSUT007
200 ppm
salicylic acid (SA) 48 4.27 µg g-1 fresh weight
96 phenolic compound 24 2.35 µg
gallic acid equivalent/mg dry mass 96
K
Mn Fe Ca

E. carotovora pv. carotovora

Marten (1999) B. subtilis B2g
spore suspension seed treatment
R. solani F. oxysporum

1)
 19

B. subtilis CaSUT007 :
:
:
:
B. amyloliquefaciens KPS 46 : BCA
SMV
SCLV ; PGPR
: BCA
; PGPR; ISR
: BCA

B. licheniformis KS217 : Pseudomonas

Xanthomonas
Bacillus sp. YP28 : ,
,
1/
BCA = , PGPR = ISR =
: ; 2552

2.5.2 (synthetic resistance inducer)

2.5.2.1 Benzo (1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BTH)

SAR catalase ascobate peroxidase
enzyme SA (Wendehenne et al., 1997)
BTH
phytoalexin biosynthetic enzymes, proteinase inhibitors,
hydrolytic enzymes, antiviral factors pathogenesis related (PR) proteins (Eikemo et al.,
 20

2003) Anfoka, G. H. (2000) BTH 7 CMV-Y

12.5
peach fruit BTH
Penicillium expansum phenylalanine
ammonialyase (PAL), polyphenoloxidase (PPO) peroxidase (POX) (Liu et al., 2005)
10

broomrape (Orobanche cumana) (Buschmann et al., 2005)

2.5.2.2 salicylic acid, SA
phenylalanine phenylalanine ammonia lyase (PAL) SA

SA SA
catalase
H2O2 SA H2O2
hypersensitive cell death
Siddiqui and Shaukat (2003) P. aeruginosa CHAO
SA
2557 200
ppm B. subtilis CaSUT007

2.5.2.3 thiamine B1 Thio-

B
pyrophosphate (TPP), coenzyme
thiamine P.
viticola HR P. viticola Boubakri et
al., 2012 ß-aminobutyric acid P. viticola
Cohen, 2002
2.5.2.4 jasmonic acid, JA)

linolinic acid lipid membrane

 21

JA phloem JA
R gene (Wasternack et al., 2006)
2.5.3 (natural resistance inducer)

N-acetyl-D-glucosamine 50

( , 2551)
receptor)

flagella)
MAMPs (microbe-associated molecular pattern) PAMPs (microbe-associated molecular
pattern) Hamel and
Beaudoin, 2010)
(elicitor) SAR

-
0.1 ROS
phenolic) secondary metabolite Iriti et al. (2010) -

Burkhanova et al., 2007)

-
 22

( , 2556)

2555 1.1-1.6
Pythium palmivora
-1
40 mg L

2.6
2.6.1

functional group) organic compound)

spectrophotometry)
enzyme linked immunosorbent assay (ELISA) UV-VIS spectroscopy
,
2551
,
, 2554
SA
, 2552 SA
, 2555

, 2555 ) (2557)
salicylic acid (SA) phenolic compound
CaSUT007 200 ppm
salicylic acid (SA) 48 4.27 µg g- fresh weight
96 phenolic compound 24
2.35 µg gallic acid equivalent/mg dry mass 96
2557 SecCaSUT007
 23

salicylic acid (SA), phenolic compound (PC) lignin P.

viticola 24 SecCaSUT007 SA
113.28±0.94 115.83±0.55 2551

4 KPS46
IAA, surfactin lipopeptide extracellular protein

X. axonopodis pv. glycines 4

KPS46 defense related enzymes
-1,3-glucanase peroxidase activity -
2558 P. fluorescens
high-performance liquid chromatography
(HPLC) IAA
IAA 3.54 µg mg-1
7 Kohler (2002) 100 Arabidopsis
3 P. syringae pv. tomato
PR-1
2.6.2 infrared
(IR) spectroscopy
infrared spectroscopy

vibration) 2
stretching) bending) 2.5-25
-1
4000-670 ( , 2547; , 2547)
Wave Number
IR Spectrum
molecular fingerprint

Functional Groups
 24

wave numbers 4000-400 cm-1

4000-1500 cm-1
hydroxyl (-OH) methyl carbonly (-CO) ( 6)
IR spectroscopy
Infrared micro spectroscopy
spectroscopy

non-destructive)

ionization)

, 2555

IR
Amide I, Amide II, lipid, carbohydrate

FITR

PGPR B. subtilis CaSUT007 (Buensanteai et al.,

2012) PGPR
polysaccharide amide I
indole-3-acetic acid high-performance
liquid chromatography (HPLC) B. subtilis
SUT007
2557) Fourier transformed infrared spectroscopy

B. subtilis CaSUT007
 25

C-H stretching C=O ester amide I

C-H bonding, C-O stretching polysaccharide

2557)
FTIR
SecCaSUT007
lipid , C=O ester, amide protein, C-H bending, cellulose, hemicellulose carbohydrate
S.
ampelinum C=O ester
amide protein
phenolic compound PR protein

C-H bending
ethylene
carbohydrate carbohydrate
SAR 2
 26

6
Salman, 2010

6
1. 4000-3100 cm-1 OH (3400 cm-1 ) NH
Stretching mode (Amide A ~ 3300 cm-1 Amide B ~ 3030 cm-1 )
2. 3100-2800 cm-1 C-H Stretching vibration CH3 CH2

3. 1800-1500 cm-1 Amide I Amide II

secondary structure alpha-helix , beta-sheet,
turn C=O ester group
4. 1300-1500 cm-1 C-H bending vibration CH3 CH2
Stretching vibration COO- Amino acid side
chains
5. 1230 cm-1 P=O asymmetric stretching vibrations
phosphodiester, free phosphate monoester phosphate
DNA/RNA Polysaccharide backbone structures
 27

6. 1200-900 cm-1 PO2- nucleic acid C-OC

C-O-P stretching vibrations oligo-polysacharide

2
Wavenumber (cm-1) Assignment
~ 3307 N-H and O-H stretching vibration : polysaccharides, protein
~2959 CH3 asymmetric stretch :mainly lipids
~2927 CH2 asymmetric stretch: mainly lipids, with the little contribution from
proteins, carbohydrates, nucleic acids
~2876 CH3 symmetric stretch: mainly proteins, with the little contribution
from lipids, carbohydrates, nucleic acids
~2857 CH2 symmetric stretch: mainly lipids, with the little contribution from
proteins, carbohydrates, nucleic acids
~1739-1744 Ester C=O stretch: lipid, triglycerides
~ 1700-1600 Mainly (C=O) associated with protein as the amide I band
~1631 ß-sheet protein secondary structure
~1653 -helix protein secondary structure
~1682 Turn
~1541 Amide II (protein N-H bend, C-N stretch)
~1452 CH2 Bending: lipids
~ 1391 COO- symmetric stretch: amino acid side chains, fatty acids
~1236 Asymmetric stretching: mainly nucleic acids with the little
contribution from phospholipids
~1152 CO-O-C asymmetric stretching: glycogen and nucleic acids
~1080 PO- 2 symmetric stretching: nucleic acids and phospholipids
 28

2.7 B. subtilis
Bacillus

Bacillus
2.7.1
Bacillus sp.
(seed
coating)
polymer

(Copeland and McDonal, 1995; , 2540 ; , 2550)

(rhizosphere)
(seed treatment)

(El-Hassan and Gowen, 2006)

Manjula and Podile (2005) B. subtilis
AF1 (peat) (peat+chitin)
Aspergillus niger (peat+mycelium) (spent compost) (alginate)
4

29 30 2 32 33

20-40 100
El-Hassan and Gowen (2006) B. subtilis
carboxymethyl cellulose (CMC)
Fusarium oxysporum f.sp. lentil 2
 29

Precoz Idlib-2

33.3
93.33
6.67 26.67
Marten (1999) B. subtilis B2g
spore suspension seed treatment
R. solani F. oxysporum

2550
SP009S SP007s
8® )
22.95, 19.03, 14.34
E.carotovora subsp.
carotovora 17.30
1.70
2.7.2

(2550)
B. subtilis 5 DOA-WB1, DOA-WB2, DOA-WB3,
DOA-WB4 DOA-WB5 R.
solanacearum 10 B. subtilis
109 cfu.ml-1 2-3 R. solanacearum 106
cfu.ml-1 B. subtilis 106 cfu.ml-1
10 4 7 5
23.4-80
DOA-WB4
15.8-44.9
DOA-WB4
 30

2545)
Bacillus sp. Pseudomonas sp.
X. oryzae pv. oryzae
27-34 69
2.7.3
EI-Hassan
Gowen (2006) 2
2 F. oxysporum f.sp. lentil
6 -1
2.5x10 cfu.ml 60 / B.
subtilis 2-3x108 cfu.ml-1 60 /
48 73
30 control 100

Metasebia et al., (2009)

BioNem B. firmus 0.5,
1.0, 1.5, 2.0 2.5 Meloidogyne incognita
(egg mass)
3
BioNem M. incognita 3
3 98 100
1,200 M.
incognita 1,000 / BioNem 1, 2, 4, 8 16
53 85
10 20 16 /
M. incognita
8 /
2.7.4
(2550) (carrier) B.
amyloliquefaciens KPS46
X. axonopodis pv.
glycines
 31

B. amyloloquefaciens 8.85x1014 cfu.ml-1

60
1.29x1013 1.03x1013 cfu.ml-1

30 20 / ( 1 : 1 )
24 X. axonopodis pv. glycines 108 cfu.ml-1
20 /
33.44, 35.83, 36.33 36.78

33.33 34.00
(2552) B. amyloliquefaciens DGg13
2 2 200
48 700 1
6 2.5x1011
cfu.ml-1 (4x1011 cfu/ )
6 2x108 cfu.ml-1
6.3x1012 cfu.ml-1
Colletotrichum spp. 86.97 84.99

2.8 B. subtilis
Bacillus

Bacillus
2.8.1 (liquid formulate)
 32

reactivation
(2538) B.
subtilis
Phytophthora sp.

1 1 -20
-150 -196

2.8.2 (dry formulate) Paau (1988)

Rajet et al., (2003) B. subtilis,

B. pumilus B. amyloliquefaciens Auburn

2552 B. subtilis CH4 1 B. subtilis

+ + B. subtilis 1.88 log cfu.ml-1
1 88
240 / Vidhyasekaran (1995)
P. fluorescens 1
pH 7.0 240
3.75x108 1.3x108 cfu.ml-1 1999
30
50 9x108 cfu.ml-1 (2538)
B. subtilis KK-TO3 CH6 B. subtilis
 33

KK-TO3 8.8x108 CH6 7.1x108 cfu.ml-1

180 104-106 4
8.8x107 6.4x107 1
Lewis (1995) Gliocladium virens
G1-3 G1-21 Trichoderma hamatum TRI-4
5 6
7 6
G1-3, G1-21 TRI-4 1.8x10 , 6.4x10 1.2x106 1.3x107, 2.6x106
3.7x104 cfu.ml-1
Kloepper (1980) Pseudomonas spp.
20 109
8.2x107 cfu.ml-1 Walker (1983)
Phyllosticta sp. clay 10-20 1.33
5x109 cfu.ml-1 4
10 1.8x107 cfu.ml-1

2.9

B. subtilis P. fluorescens Top

A506 Stretomyces griseoviridis Mycostop

Bacillus
 34

Bio-Fungus®, Binab T®, Rootshield®

Bio-Fungus®, Binab T®, Rootshield®, T-22 Planter Box® pelleted
Binab T® Promote® (Haggag et al., 2011) ( 3)

3 Bacillus

Bacillus spp.

Serenade B. subtilis QST 713 WP Fungi, bacteria on multiple

Aqueous suspension vegetables, fruits
EcoGuard B. licheniformis SB3086 Flowable Sclerotinia homoeocapa on
turf
Kodiak B. subtilis GB03 WP (conc.)1 Fungi on cotton,
Flowable large-seeded
Yield shield B. pumillus GB34 WP (conc.) Fungi on soybeans
Bio yield B. amyloliquefaciens B99 Dry flake Fungi on bedding plants in
+ B. subtilis GB122 potting mixes
Subtilex B. subtilis M1600 WP (conc.) Fungi on cotton, large-
seeded legumes, soybeans
Hi stick+ B. subtilis M1600+ Flowable Fungi on soybeans, peanut
Subtilex Rhizobium
: Schisler (2004); 2556
1
wetlable powder concentration

2.10 spray drying)

spray drying)

Risch, 1995; Qi and Xu, 1999)

, ,
 35

flowability), bulk density),

dispersibility), (Augustin and Sanguansri,
2008)
1930 Dziezak, 1988)

feed)

drying chamber)
atomizer

, 2551)

Dziezak, 1988) Tsvetkov (1983) Micrococcus

varians M95 Lactobacillus plantarum L4
5

Kim and Bhowmik

(1990) S. thermophilus L. bulgaricus
outlet air temperature) 2
inlet air temperature) atomizing air
pressure Gardiner et al. (2000) L. paracasei
 36

NFBC 338 170 70-120

80-85 L. paracasei NFBC
338 4.1

2555
25 ml/min 15 kg/cm
65, 75 85 L. fermentum
2311M 45.0, 32.4 15.5 L. casei
sub.sp. paracasei F-19 21.5, 10.9 2.7
L. fermentum
2311M L.casei sub.sp. paracasei F-19 85
3.2 3.5 75
5.2 5.5
65 7.9 8.1
(2552) 130
70 L. plantarum KMITNB 53.4 maltodextrin 10
+monosodium glutamate (MSG) 10 spray drying carrier
17 (8.29 ± 0.07 log cfu.ml-1)
Graham (1998)
Arthrobotrys dactyloides A4 GCS
10,000
10 vermiculite kaolin
electrically powered pasta maker 1
4-5 2
0.5 2.5 4
18 A. dactyloides
Meloidogyne javanica
gall 90
 37

Lam (2001) poly lactic-co-glycolic acid (PLGA) recombinant

human nerve growth factor (rhNGF) rhNGF

Streptococcus
spp. 50-80

yeast extract
L. bulgaricus
5
7
Streptococcus sp.

L. plantarum
3.8-5.7 0.7-2.3

, 254)

2.10.1
 38

2.10.1.1

Foster (1962) stationary phase

Heckly (1978) 24
3-7 Espina Packard
(1979)
neutralization)
stationary phase
Teixeira
1995) L. bulgaricus inlet air temperature)
200 outlet air temperature) 80
stationary phase exponential
phase heat shock exponential phase
heat shock heat shock
stationary phase 2555
B. subtilis B006 NB glucose 2
B. subtilis B 48 28
6.3x10 cfu.ml-1 B. subtilis B076
96 28 1.4x10 cfu.ml-1 BRS038
48 32 36
5 4x10 cfu.ml-1
2.10.1.2

Kilara 1976) L. bulgaricus

6x105 cfu.ml-1 7x104 cfu.ml-1 75
10 L. acidophilus 1.6x105 cfu.ml-1
 39

2.6x104 cfu.ml-1 63 19
S. thermophilus 7.3x10 cfu.ml-1
5
2.5x10 cfu.ml-1
4

2.11

2.11.1 (carrier)
vermiculite, clay, calcium sulphate mineral soil

Fravel
(1995)
96.6, 25.4 21.1
Talaromyces flavus
Lomer (1993)
kerosene, neem-oil
Metarhizium flavoviride 5x1010 cfu.ml-1 4x109 cfu.ml-1
3-4 . .1995 Douro-kpindou kerosene peanut oil 1:1
9 -1
M. flavoviride 2x10 cfu.ml
Zonocerus variegatus Mark (1996) diatomaceous earth
C. truncatum 2x106 cfu.ml-1 airdired
4 18 93
18
80
2.11 2 (binder)

Davis (1992)
 40

Chaetomium
globosum flyspeck Zygophiala jamaicensis
2.11 3 (protectant)

alpha, alpha-trehalose-borate L. acidophilus

trehalose
borate trehalos
( , 2535)
4
Takeuchi (2000)
-

5, 10 10
0.2 30-40
Abadias (2001) 10
-20
Candida sake 28.9
Champagne (1991)
Streptococci thermophilus
Streptococci 10 20
Gardiner 2000 170
80
L. paracasei NFBC 338 L salivarius UCC 118 97
11 Cytoplasmic membrane
 41

4 Bacillus sp.

Liquid carriers Vegetable oils

Mineral carriers Kaolinite clay, diatomaceous earth
Organic carriers Grain flour
Stabilizers Lactose, sodium benzoate
Nutrients Molasses, peptone
Binders Gum Arabic, carboxymethyl cellulose
Desiccants Silica gel, anhydrous salts
Thickeners Xanthan gum
Surfactants Tween 80
Dispersants Microcrystalline cellulose
: Schisler (2004); 2555

2.12 B. subtilis

(starter culture)

(inoculum)

2 1)
2)
 42

Rhizopus

2.13

2 1) (physical
method) 2 (chemical method)
2.13.1 (physical method) 3
1. (high temperature) (dry heat)
(moist heat)
2. (radiation)
3. (filtration)
2.13.1.1 (dry heat)
2.13.1.1.1 incineration)
loop, needle, swab

loop

2.13.1.1.2 (hot air oven)

160-170
1-2
160
2 steam
dry heat sterilization
dry heat sterilization
 43

2.13.1.2 moist heat)

2.13.1.2.1 (boiling) 100
10
20

Boiling water
10 100
20 100

2.13.1.2.2 (pasteurization)
Louis Pasteur) 100

2.13.1.2.3 Sterilization) 100

Ultra high Temperature: UHT)

135-150 1-4
6
2.13.1.2.4 autoclave) Steam
under pressure
15 121 15

steam sterilization
gravity or downward displacement pre-vacuum flash or high speed
 44

flash or high speed 132

27 (exposure time) 3-10

implant

2.13.1.3 (radiation)

ionize DNA
ionizing radiation
200 nm
syringe

non ionizing radiation TB 5

130-400 nm
260 nm

2.13.1.4 filtration)

Osmosis

, 2550
2.13.2 (chemical method)

2
2.13.2.1 Preservatives)
, ,
 45

2.13.2.1.1
Antioxidants) BHA BHT Antibrowning)

Bacteriocin) Lactobacillus sp., Pediococcus sp.

Lactococcus sp.

(chemical method)

sterilants
disinfectants antiseptic
chemical method

alcohol 60-90 70
sodium bisulfite
yeast) mold) bacteria)
wine) 0.01-0.02 ( -
, 2554) hydrogen peroxide 30
disinfectant 6 sterilant
sulfite potassium metabisulfite, PMS
sulfur dioxide, SO2

0.1-0.15 , 2550
KMS 5
 46

5 PMS

dry red wine) 160

dry white wine) 210
5 / 210
5 / 260
: , 2546; , 2550
 3

3.1 B. subtilis
3.1.1 B. subtilis
B. subtilis CaSUT007 stock

4 1 molasses and diamonium phosphate MD 2 molasses diamonium

phosphate and yeast extract (MDY) 3 molasses M
nutrient glucose broth (NGB) CRD 3
3 120
15 / 20 B. subtilis CaSUT007
1 loop rotary shaker 120 /
28-30 48
dilution plate count 8 10-1 - 10-8
(spread plate) nutrient agar (NA) 3
24 30-300
;
2558, Nancy et al., 2011

cfu.ml-1) = ×
(ml)

3.1.2 B. subtilis
spray dry)
2 1
carboxymethyl cellulose (CMC) 2 carboxymethyl cellulose
(CMC) 10:1 ( , 2552; Teera-arunsiri, 2003)
CRD 2 3
 48

120 15 / 15 2
1
3.1.1 48 2
5:1 spray dry
3 80, 75, 65
0.1
dilution plate count 3.1.1
3.1.1
3.2.1 ( , 2555; Franks et al., 1991; Meng, 2015)
3.1.3 B. subtilis

B. subtilis
3.1.2 26-30

6 dilution plate count

3.1.1 3.1.1
( Prescott, 2002;
Wiyono et al., 2008)

3.2 B. subtilis
3.2.1
CRD starter 3
1 MDY 2 1 10 1 3
MD (molasses 25 , diamonium phosphate 1 1 )+ 1
3 ¼
120 15 /
15 B. subtilis CaSUT007

(O.D.) 600 dilution plate count

 49

5 0, 0.1, 0.2, 0.3,

0.4 0.5 1

3 1 6, 12, 18 24
rotary shaker 100 / 1 2
2 12 24 3
24 ( , 2529)
3.2.2
3.2.2
(starter) (multiplication media)

CRD 3
3.2.1
5 1) 121 15
/ 20 2 30 3)
(PMS) 150 ppm, 200 ppm 250 ppm 6
24 4) (SMS) 150 ppm, 200 ppm 250 ppm
6 24 5.)
dilution plate count 3.1.1

starter culture
3.2.1
: 1:10,
1:100, 1:200 2 24
dilution plate count , 2542;
, 2542; Wiyono et al., 2008
3.2.3

B. subtilis
CaSUT007 2
 50

121 15 / 20
molasses diamonium phosphate and yeast extract (MDY)
3.2.2
12
B. subtilis CaSUT007
1:10 24 2
dilution plate count 3.1.1
, 2553)
3.2.4

1:5 120 15 /
20 B. subtilis CaSUT007
1 loop rotary shaker 120 /
28-30 24 NGB
dilution plate count 3.1.1
B. subtilis CaSUT007
, 2542; , 2542
3.2.5

factorial in CRD 3
10 2 1
: 5 1:50, 1:100, 1:150, 1:200
2 4 1) 2) 3)
4)
15-20 10
0.5 1 2 suspension E. carotovora pv. carotovorum
O.D. 0.2 x cfu.ml-1) 5
(disease severity) 7
0-4 0 = , 1=
25% ,2= 25-50% , 3= 50-75% , 4=
 51

75% , 2549; , 2557; Vincelli

and Hershman, 2011)
, 2557; Vincelli and Hershman, 2011
=( / x 100
% = [(A-B)/A] ×100
A=
B=

0 5

3.2.6
Randomized Complete
Block Design (RCBD) 3 10

1 3.2.4 1
10
2 3.2.4 2
10 20
3 3.2.4 3
10, 20 30
4
3.2.4 1 10
5
3.2.4 2 10 20
6
3.2.4 3 10, 20 30
7 8
10
8

9
 52

45 E. carotovora pv.
carotovorum suspension NGB 100
72 150 /
108 cfu.ml-1
spectrophotometer 600 O.D. 0.2 20
3 (disease severity )
(disease incidence) 7
3.2.4 , 2549;
, 2557; Vincelli and Hershman, 2011)
, 2549; ,
2557; Vincelli and Hershman, 2011)

=( / x 100
% = [(A-B)/A] ×100
A=
B=
0 5

3.2.7 FTIR
microspectroscopy
3.2.7.1

60 48
Infrared (IR) Spectrometer
, 2555; , 2010; , 2557;Buensanteai
et al., 1012)
3.2.7.2 FTIR microspectroscopy
3.2.6.1
FTIR microspectroscopy
fourier transform infrared spectroscopy:FT-IR (Tensor 27, Bruker optic) attenuated
total reflectance (ATR) mercury cadmium telluride (MCT)
 53

600-4000 cm-1 Resolution 6 cm-1 scan 64

unscrambler X 10.1
(CAMO, Norway) multination data analysis principal component analysis (PCA)
( , 2555; , 2555; , 2557;
, 2557;Buensanteai et al., 2012;Thumanu et al., 2014)

3.3
SPSS for windows
v.14.0
multiple range test (DMRT)
 4

4.1 B. subtilis
4.1.1 B. subtilis
B. subtilis CaSUT007
3 48 nutrient glucose broth
(NGB) molasses diamonium
phosphate and yeast extract MDY) 1.30±0.16x109 cfu.ml-1
NGB 1.27±0.15x109 cfu.ml-1
molasses and diamonium phosphate (MD)
1.43±0.27x107 cfu.ml-1 molasses (M 2.37±0.20x106
cfu.ml-1
MDY NGB 4.1 4.1)
MDY NGB

4.1 B. subtilis 4 48

(cfu.ml-1)1/
Molasses and diamonium phosphate 1.43±0.27x107b
Molasses diamonium phosphate and yeast extract 1.30±0.16x109a
Molasses 2.37±0.20x106b
Nutrient glucose broth 1.27±0.15x109a
F-test **
CV (%) 5.84
1/
cfu.ml-1) = ×
(ml)
 55

A B C D

E F G H H

4.1
100X B. subtilis CaSUT007 A
E) molasses and diamonium phosphate MD B F)
molasses diamonium phosphate and yeast extract (MDY) C G)
molasses M D H) nutrient glucose broth (NGB)

4.1.2 B. subtilis
Spray dry)
MDY B. subtilis
4.1.1 2
MDY 1
CMC 2 CMC
2 9:1
3 65, 75 80
2
4.2 1

1 75 65
1.40±0.14x108 1.13±0.23x108 cfu.g-1 80
 56

1.63±0.11x106 cfu.g-1

1.21x109 cfu.ml-1
2
75 65 1.17±0.91x109 1.15±0.21x109
cfu.g-1 x cfu.ml-
80
2.36±0.12x106 cfu.g-1

2
CMC 75
65

A B C

4.2 B. subtilis CaSUT007 2

spray dry A) 80
B) 75 C) 65
 57

4.2 B. subtilis 2
3
B. subtilis (cfu.g-1)1/
12/ 23/
80 1.63±0.11x106b 2.36±0.12x106b ,
75 1.40±0.44x108a 1.17±0.91x108a ,
65 1.13±0.23x108a 1.15±0.21x108a ,
F-test ** **
CV (%) 5.20 6.35
1/
N= , DF = dilution facter, V0 =
strok , Va = spread plate l , W0 =

2/
1 CMC
3/
2 CMC

4.1.3 B. subtilis
2
4.1.3.1
26-30
2 2
75
6 dilution plate count
0 2 2.14×108, 1.12×108
1.08×108 cfu.g-1 3-6 9.6×107,
1.04x107, 9.6x107 1.19x106 cfu.g-1
4.3 4.3
 58

4.3 B subtilis CaSUT007 spray dry

2 6 2
B. subtilis (cfu.g-1)1/
0 2.14±0.16×108a
1 1.12±0.30×108a
2 1.08±0.65×108ab
3 9.6±0.11×107b
4 1.04±0.41x107ab
5 9.6±0.37x107b
6 1.19±0.75x106c
F-test **
CV (%) 10.86
1/
N= , DF = dilution facter, V0 =
strok , Va = spread plate, W0 =

A B C D

E F G

4.3 B. subtilis CaSUT007 spray dry

2 A) B) 1 C) 2 D)
3 E) 4 F) 5 G) 6
 59

4.1.3.2

2 75
6
2.92, 5.65, 7.09,
9.31, 11.53, 9.99 8.07
1.45, 1.18, 2.46, 3.1, 2.24, 1.37 1.67 4.4

4.4 B. subtilis
CaSUT007 2 6
1/

0 2.92 1.45
1 5.65 1.18
2 7.09 2.46
3 9.31 3.1
4 11.53 2.24
5 9.99 1.37
6 8.07 1.67
1/
( , 2553 )

4.2 B. subtilis
4.2.1

3 B. subtilis
CaSUT007 6
1 2 MDY
3 MDY 3
 60

2 MDY 1.0×108 cfu.ml-1

(OD=0.207) 3 + MDY 1
5.0×107 cfu.ml-1 (OD=0.199) 2.60×107cfu.ml-1 (OD=0.190)
4.5
B. subtilis
CaSUT007 5 0.1, 0.2, 0.3,
0.4 0.5 1
5
0.5 1.7×108 cfu.ml-1 (OD=0.215)
0.4, 0.3, 0.2 0.1 1.5×108, 6.5×107,
9.0×106, 1.5×107 cfu.ml-1 (OD=0.214, 0.203, 0.177 0.185) 4.6

B. subtilis CaSUT007 3 1.
6 24 2. 6 12 3.
3
1
8.2×107cfu.ml-1 (OD=0.208) 2
3 4.1×107, 2.60×107 cfu.ml-1 OD=0.198
0.190 4.7
MDY
0.5 1
6

1
49 19 0.8
 61

4.5
B. subtilis CaSUT007

(A 600) cfu.ml-1 2/
1 0.190c1/ 2.6×107
2 MDY 0.207a 1.0×108
3 + MDY 0.199b 5.0×107
F-test **
CV (%) 15.46
1/
95% P 0.05
DMRT
2/
Absorbance 600 nm (Log of cell (cfu.ml-1))
1

4.6
B. subtilis CaSUT007

(A 600) cfu.ml-1 2/
0 0.03d1/ 0
0.1 0.185c 1.5×107
0.2 0.177c 9.0×106
0.3 0.203b 6.5×107
0.4 0.214a 1.5×108
0.5 0.215a 1.7×108
F-test **
CV (%) 20.64
1/
95% P 0.05
DMRT
2/
Absorbance 600 nm (Log of cell (cfu.ml-1)
1
 62

4.7
B. subtilis CaSUT007

(A 600) cfu.ml-1 2
6 0.208a1/ 8.2×107
2 0.198b 4.1×107
0.190c 2.6×107
F-test **
CV (%) 18.72
1/
95% P 0.05
DMRT
2/
Absorbance 600 nm (Log of cell (cfu.ml-1))
1

4.2.2
starter) multiplication media)

dilution plate count

121 15 /
20 (PMS) 250
ppm 6
1±0.14×101 cfu.ml-1 200 ppm 150 ppm
9.3±0.72×105 7.4±0.34×107 cfu.ml-1

4.8
4.4
starter)
multiplication media) 0.3
MDY 1 3
1:10, 1:100, 1:200
 63

(PMS) 250 ppm 1:10 1.47±0.86×108 cfu.ml-1

1:100, 1:200 1.86±0.13×106 1.64±0.19×106
cfu.ml-1
121 1:10, 1:100,
1:200 1.30±0.18×108, 1.25±0.81×106 1.39±0.13×105 cfu.ml-1
4.9 4.5

4.8
2/
(cfu.ml-1)1/
Autoclave
1.22±0.58×108c
PMS 150 ppm, 6 hr. 7.4±0.34×107bc
PMS 200 ppm, 6 hr. 9.3±0.72×105 bc
PMS 250 ppm, 6 hr. 1.0±0.14×101a
PMS 150 ppm, 24 hr. 4.6±0.63×108cd
PMS 200 ppm, 24 hr. 9.8±0.11×107bc
PMS 250 ppm, 24 hr. 1.1±0.06×104 ab
SMS 150 ppm, 6 hr. 8.0±0.60×108 cd
SMS 200 ppm, 6 hr. 1.56±0.19×108c
SMS 250 ppm, 6 hr. 1.24±0.27×108c
SMS 150 ppm, 24 hr. 1.43±0.12×108bc
SMS 200 ppm, 24 hr. 7.9±0.58×107b
SMS 250 ppm, 24 hr. 1.05±0.10×107b
1.53±0.11×108c
F-test *
CV (%) 19.59
1/
cfu.ml-1) = ×

2/
PMS
SMS
Autoclave 120 15 / 20
100 10
 64

A B C

150 ppm 200 ppm 250 ppm

PMS 6

D E F

PMS 24

G H I

SMS 6

J K L

SMS 24

M N O

4.4 A)
Control B) 121
C) 30 D-F) PMS 150 200
250 ppm 6 G-I) PMS 150
200 250 ppm 24 J-L) SMS
150 200 250 ppm 6 M-O) SMS
150 200 250 ppm 24
 65

4.9 starter)
mortification media)
B. subtilis (cfu.ml-1)1/
Autoclave, 1:10 1.30±0.18×108b
Autoclave, 1:100 1.25±0.81×106c
Autoclave, 1:200 1.39±0.13×105c
PMS 250 ppm, 1:10 1.47±0.86×108a
PMS 250 ppm, 1:100 1.86±0.13×106c
PMS 250 ppm, 1:200 1.64±0.19×106c
F-test **
CV (%) 5.5
1/
cfu.ml-1) = ×
(ml)

1:10 1:100 1:200

autoclave

A B C

PMS 200 ppm

D E F

4.5 starter)
multiplication media) A-C) 121
1:10, 1:100, 1:200 D-
F) PMS 200 ppm
1:10, 1:100, 1:200
 66

4.2.3

10
dilution plate count 8
10-1 - 10-8 spread plate) nutrient agar (NA)
3 24 2

2 (PMS)
250 ppm 12-24
B. subtilis CaSUT007

B. subtilis CaSUT007
1.59 × 108 cfu.ml-1
4.6

A B

4.6 molasses diamonium phosphate and yeast extract (MDY)

B. subtilis
30 24 A) B)
PMS
 67

4.2.4

B. subtilis CaSUT007 NGB

24 B. subtilis 5
4.7 B pH pH 3.3
pH 6.0-7.0 1.9
100 B. subtilis CaSUT007 3
B. subtilis CaSUT007
NGB 2.24×106 cfu.ml-1
4.7 A

A B

4.7
B. subtilis CaSUT007 A) NGB B)
 68

4.2.5

KMS 250 ppm

2 1 :
5 1:50, 1:100, 1:150, 1:200 2
4 1) 2) 3) 4)
15
11 1 50
1.26±0.23 65
12
1:100
1.30±0.21 64 13
1:150
1.33±0.21 62
4.10
 69

4.10
B. subtilis
1/ 3/
(
1:50 1.76±0.25bc2/ 49.0
1:100 1.93±0.20cd 44.0
1:150 1.90±0.24cd 45.0
1:200 2.06±0.20d 40.0
3.43±0.16e 0.0
1:50 1.80±0.20c 46.0
1:100 1.81±0.24b 46.0
1:150 1.83±0.36c 45.0
1:200 1.88±0.24b 44.0
3.33±0.16e 0.0
1:50 1.26±0.23a 65.0
1:100 1.30±0.21ab 64.0
1:150 1.33±0.21ab 62.0
1:200 1.64±0.27bc 53.0
3.52±0.14e 0.0
F-test **
CV (%) 33.53
1/
0-4 0 = , 1= 25%
,2= 25 50% , 3= 50 75% , 4= 75%
2/
95% P 0.05
DMRT
3/
 70

4.2.6

4.2.5 6
1:50 3 10, 20 30

0.79±0.33 92 5
1 50 2 10 20
1.70±0.61
70 3.96±0.29
0.0 8
1.76±0.53 69

3.96±0.29 27
4.11
 71

4.11
B. subtilis
3/ 1/ 4/

1 3.10±0.41d2/ 45.0
2 2.80±0.32cd 53.0
3 2.20±0.56c 61.0
4 2.40±0.47c 63.0
5 1.70±0.61b 70.0
6 0.79±0.33a 92.0
7 3.55±0.49e 27.0
8 1.76±0.53b 69.0
9 3.96±0.29f 0.0
F-test **
CV (%) 18.22
1/
0-4 0 = , 1= 25%
,2= 25 - 50% , 3= 50 - 75% , 4= 75%
2/
95% P 0.05
DMRT
3/
1 1:50 1 2
1:50 2 3 1:50 3 4
1:50 1 10 5
1:50 2 10 20 6
1:50 3 10, 20 30 7
8 10 8 -
9
4/
 72

A B C

D E F

G H I

4.8

E. carotovara pv. carotovora ECC 7 A) 1

1:50 1 B) 2 1:50 2 C)
3 1:50 3 D) 4
1:50 1 10 E) 5
1:50 2 10 20 F)
6 1:50 3
10, 20 30 G) 7
8 10 H) 8
30 20 I) 9
 73

4.2.7 FTIR
microspectroscopy

B. subtilis CaSUT007
(Control) FTIR microspectroscopy Vertec70, Bruker optic
OPUS 7.0 (Bruker optic, German)
Principal component analysis (PCA) The Unscrambler X (CAMO, Norway)
Second derivative 1739 cm-1
(lipid, triglycerides) 1631 cm-1 (ß-sheet protein) 1642
cm-1(amide I) 1560 cm-1(amide II) 1502 cm-1 (amide II)
1460 cm-1 (lipids) 1406 cm-1 (amino acid, fatty
acids) B. subtilis , amide
I, amide II amino acid
Second derivative 4.9 4.10
 Control
SJN007
Second drivertive of absorbance

1560
1406
1631 1460
1502
1739

Wavenumber cm-1

4.9 Second derivative B. subtilis CaSUT007 (SJN 007)

(Control) 2005-759 cm -1

74
 Control
SJN007

A B

C D

4.10 A,B,C D Principal component analysis (PCA)

B. subtilis CaSUT007 (SJN 007) (Control)

75
39
 5

Bacillus
subtilis
(inoculum)
(starter medium)
(fermenting or multiplication
medium)

1.
Bacillus subtilis MDY 48
1.30±0.16x109cfu.ml-1
2546)
B. subtilis TISTR 001 109 cfu.ml-1
(2536) B. subtilis
B31
1.2×1010 cfu.ml-1 endospore)
vegetative cell) Bacillus
endosporeforming) 1 1

Kloepper et al., 2004

2. inlet 140
(outlet) 75
1.40±0.14x10 cfu.g-1 Kim and Bhowmik (1990)
 77

S. thermophilus L. bulgaricus outlet air temperature)

2
inlet air temperature) atomizing air pressure Gardiner et al.
(2000) L. paracasei NFBC 338
170 70-120
80-85 L. paracasei NFBC 338

, 2553
B. subtilis
(Sandra and Greg et al., 2000) CMC

P. fluorescens
SP007s

(Preecha, 2009) Van Dyke

(2000) insch sandy loam soil 2.13
 78

0.19 P. fluorescens
MON787 1 x 109 cfu.ml- 25
2 2 log phase 4 log
phase 1-2 Vidhyasekaran (1995)
P. fluorescens
1 7.0 240
3.75 x 108 1.3 x 108 cfu.ml-
2 (spray dryer)
freeze dryer)

, 2558

6 1.19±0.75x106cfu.g-1

2538)
Bacillus spp. NK-TO3 CH6
P. fluorescent s F2

3
1:2:2 ( 2548)
22
4 P. fluorescens NA 1 SU1 B. subtilis
CH4 CH6
1

2553 1
9 XA6 , , CMC
60, 30, 8, 2
1.63 × 1010 cfu.ml- (28-32 ) 6
8 69-78
B. subtilis CaSUT007
 79

2.8 × 107 cfu.ml- 6

1.58 x 107 cfu.ml- 1999
30
8 -
50 9x10 cfu.ml Alan (1988)

Cavalho et al., (2004)

mannose L. buigarigus fructose, lactose

glucose Streeter et al., (2002) trehalose
Bradyrhizobium japonicum Abadias et al.,
(2001) skimmed milk carbohydrate sugars Candida sake
45-85 Desmond et al., (2002) L. paracasei
1000 Gum acacia 10 skimmed milk 10
Hamsupo (2005) skim milk 18 monosodium glutamate 2
spray drying carrier L. reuteri KUB-AC5
87.24 Champagne (1991)

Streptococci thermophilus
Streptococci 10 20 Gardiner 2000
170 80
L. paracasei NFBC 338 L.
salivarius UCC 118 97 11
Cytoplasmic membrane
5.1
 80

5.1
: Bashan et al., 2014

carrier)
amendment)

5.1) (Chisler et al., 2004) Bashan et al., (2015)

NaOH, MgSO4·7H2O, CaCl2, NaCl, NH4 Cl, yeast extract, FeCl3, NaMoO4 ·2H2O,
MnSO4, H3BO3, Cu(NO3)2·3H 2 O ZnSO 4·7H2O
Azospirillum spp. Ya ´nez et al., (2011)
skimmed milk, skimmed milk plus, MgSO , MgSO and MgSO 10, 10,
10, 10 20 B. subtilis
CPA- 1. . × cfu.g-1
 81

5.1 Bacillus spp.

Liquid carriers Vegetable oils

Mineral carriers Kaolinite clay, diatomaceous earth
Organic carriers Grain flour
Stabilizers Lactose, sodium benzoate
Nutrients Molasses, peptone
Binders Gum Arabic, carboxymethyl cellulose
Desiccants Silica gel, anhydrous salts
Thickeners Xanthan gum
Surfactants Tween
Dispersants Microcrystalline cellulose
Schisler ; 2555

3.
2 MDY
25 1
1.0×108 cfu.ml-1 (O.D=0.207) 0.5
1 1.7×108 cfu.ml-1 (O.D=0.215)
6
8.2×107 cfu.ml-1 (O.D=0.208)

diammonium phosphate DAP

DAP

, 2553

2550 B. subtilis
 82

19.85 2554
L. acidophilus TISTR
1338

2539 B. subtlilis B31 SM

B. subtlilis B31

riboflavin, pantothenic acid biotin

fish hydrolysate
MRS central composite design
(CCD) 72
MRS , 2554 2551
L.
plantarum PD110

MRS

2544) Pediococcus acidilactici M6

7 9
37 50
1.70x109 cfu.ml-1

50 60 20
Stoppok Buchholz, 1993)
2544
Pediococcus pentosaceus BT520
 83

molasses)
ami-ami solution, brewers yeast autolysate
molasses 20 1.7x1010 cfu.ml-1)
Pediococcus pentosaceus BT520
EM

DAP

DAP
1998 Yokochi et al.,
Schizochytrium sp. monosaccharides
S. limacinum
SR21 2556
PGPR molasses 2
- Azotobacter sp.
108 cfu.ml- 5 molasses , PEG, phosphate
buffer 2, 0.5 0.5 Azospirillum sp.
8
10 8
molasses, phosphate buffer 2 1.0
Stephanie et al., (2014) , CaCO3, MgSO4 7H2, MnSO4 H2O, FeSO4 7H2O
CaCl2 4H2O 10, 0.1, 0 0.00011, 0.04, 0.028 0.03
B. cereus

B. subtilis CaSUT007 B.
subtilis
 84

2559

indol-3-acetic acid (IAA), ,

,

, 2553

4.
starter) (multiplication media)

121
(PMS) 250 ppm
1 -
1.00±0.14×10 cfu.ml
0.5 MDY 1
(PMS) 250 ppm 1:10
1.47±0.86×108 cfu.ml- PMS

sterilization
 85

PMS
SMS
PMS 200 3 60
pH ( , 2525;
, 2546) PMS
wild yeast)
PMS

0.01 0.02 , 2554

2556
potassium metabisulphite, PMS) 0.08
1-2
2542) 1:3
180 0.4-0.6
50-100
diethylpyrocarbonate DPC 1 1
Macek et al.,1994 PMS
6-12 , 2556) PMS
24 PMS
6
24 PMS B. subtilis
CaSUT007 6-12 B. subtilis
5.

B. subtilis CaSUT007 1:50

3 10 10, 20 30

0.79±0.33 92

(copper
hydroxide) 69
 86

3.96±0.29 27 B. subtilis

secondary metabolites B. firmus KPS44 KPS46

secondary metabolites X. axonopodis pv.glycines
( , 2548)
B. subtilis
2549 P. fluoresens SP007s B.
licheniformis SP009s secondary metabolite
E.carotovora subsp. carotovora, X. campestris pv. campestris, Pythium sp.,
Alternaria sp.
X. campestris pv. campestris
(Sally et al.,
2002) Alternaria sp.
( , 2545)

4
/
2

(Crump, 1998)

(2548) 3
B1228 B1317 B1348 X. axonopodis pv. dieffenbachiae

81-89 B. amyloliquefaciens KPS P. pabuli

SW01/4 Alternaria sp.
43.0, 52.0 45.0
 87

91.0, 45.5 39.8 30.8 Prathuangwong et al.,

2004, Prathuangwong et al., 2006a; Prathuangwong et al., 2006b)
2551 SP009s
6 -
1.22 × 10 cfu.ml (O.D.=0.15) 4 SP007s 2.3×106 cfu.ml-
(O.D.=0.1) 14, 28, 42 56
68.4 80.3
2557 )
CaSUT007 200 ppm

2559 B. subtilis TU-Orga1

TU-
Orga1 X. campestris pv. campestris Alternaria sp.
5.0-5.1 80.0-83.3 Berger et al. (
B. subtilis
6. FT-IR
microspectroscopy 1642 cm-1(amide I)
1739 cm-1 (lipid, triglycerides) 1560 cm-1(amide II)
1502 cm-1 (amide II) 1460 cm-1 (lipids)
1406 cm-1 (amino acid, fatty acids) Bacillus
CaSUT007 compound lipid)
phospholipid), glycolipid) (lipoprotein)
proteins)

cellulose), hemicellulose),
(chitin), lignin) pectin) ( , 2545)
Bacillus
CaSUT007
 88

nonspecific elicitors)
glycoprotein) protease, cutinase,
cellulose hemicellulose

cellulose hemicellulose
hemicellulose (secondary cell wall)
Hernández-Blanco et al., 2007; Eggert et al., 2014; Nafisi et al., 2014)
cellulose hemicellulose ROS
phytohormones Vickers et al., 2009; Loreto et al., 2001)
C-H bending
ethylene isoprene
ISR
cell membranes Sweeting,
1996; Monson et al., 2013; Eggert et al., 2014)
2557) Fourier transformed infrared spectroscopy

Bacillus CaSUT007 C-H

stretching C=O ester amide I
C-H bonding, C-O stretching polysaccharide

2557)
FT-IR
SecCaSUT007 lipid , C=O ester,
amide protein, CH bending, cellulose, hemicellulose carbohydrate
S. ampelinum
C=O ester
amide protein
phenolic compound PR protein
C-H
bending ethylene
 89

carbohydrate carbohydrate
SAR ROS PR protein
HR FTIR
microspectroscopy

interval times
7.

B. subtilis CaSUT007

Jiamwijit,
2002)
 90

5.2
 91

5.2
: Bashan et al., 2014
 92

B. subtilis P. fluorescens Top

A506 Stretomyces griseoviridis Mycostop ( , 2556)
Minuto 2006) Mycostop® S.
griseoviridis K61 Sphagnum peat
S. griseoviridis K61
Mycostop® (Fusarium oxysporum
®
f.sp. radicis-lycopercici, F. oxysporum f.sp. lycopersici) Mycostop

Bacillus

Bio-Fungus®, Binab T®, Rootshield® Bio-Fungus®,

Binab T®, Rootshield®, T-22 Planter Box® pelleted Binab T®
Promote® (Haggag et al., 2011) Srinivasan (2009)
plant growth promoting
microbial consortia: PGPMCs) sunflower
necrosis virus
PGPMC-1 talcum B. licheniformis strain MML2501+
Bacillus sp. strain MML2551+P. aeruginosa strain MML2212+S. fradiae strain MML1042
40.9

5.3
 93

5.3
: Bashan et al., 2014
 94

5.5

10

A B

C 1

B.subtilis CaSUT007
6 24

1 10

5.4
B.subtilis CaSUT007
 95

5.5 B.subtilis CaSUT007

B. subtilis

B. subtilis
CaSUT007
 . 2524 . . : . .
. (2556). Bacillus spp.
. . 99
2552 -
Ralstonia solanacearum -
90 .
(2555).
. .
, . (2542).
Aspergillus parasiticus 102566
.
37. 3-5 2542.
. (2559). Bacillus subtilis TU-Orga1
. . 24 (5): 793-
812.
. (2554). L(+) Pediococcus
pentosaceus SW4-3 .
49. . 281-288. 594 .
2010). FT-IR spectroscopy micro
scope
http://www.technology media.co.th/articledetail.asp?arid=6072&pid=467.
2555). FTIR Spectroscopy Microscope
Technology. 37(212): 95-96.
. (2553). [ ]. :
http://kanyarat2542.blogspot.com/2010/11/blog-post_681.html.
2553 .[ ]. :
http://www.agriqua.doae.go.th/forecast/week/week53/160853softrot/Bacterialsoftrot.html
 97

. (2559). .[ ].
: http://www.thairath.co.th/content/635192
. (2558).
. .
. 2557). Erwinia carotovora pv.
carotovora .
95
, 2550 -
Bacillus amyloliquefaciens KPS46 .
8. .
(2537).
912
(2538). Antagonist Erwinias
. 111
2556 . PGPR .

2549-2550
(2551). Bacillus amyloliquefaciens KPS46

. 148 .
2555).
IR. .
http://www.slri.or.th/th/index.php?option=com.
. (2555). [ ]. : http://www.thaikasetsart.com/
. (2556). [ ]. :
http://www.thaikasetsart.com/
. (2554) .[ ]. :
https://surathai.wordpress.com/2011/05/15/fruitwine/.
, (2552).
 98

9.
24-26 2552.
2555).
1
13.
(2557). Bacillus subtilis CaSUT007

120
. 2544 . .
. .
(2556).
1(2): 81-88.
. (2552). Pseudomonas fluorescens SP007s
salicylic acid .
46. . 611-620.
, , . 2535
Bacillus subtilis. 10: 85-89.
2547).
305-344.
2550 .
. (2558). Bacillus subtilis)
.

. .
, . (2525).
.
20. 88-
100.
, , 2548).
 99

Ralstonia solanacearum. -
43. 240-247.
. (2553).
. . 122
, 2554).
42(2)
( 385-388.
, (2551) . -
Pichia pastoris YW-11430 Pichia pastoris YM-11430

46. 278-285
.(2545). .
. .
2556 Streptomyces griseus subsp. formicus

112
2551
2551.

. (2539). . . .
2555 .

22 . 1-6.
. (2556). -
[ ]. : http://personal.sut.ac.th/
montarop/2013%20WBSITE/School of_Biotech/Blog/Entries/html.
. (2553). .
.
(2546). Bacillus subtilis
TISTR 001 [ ]. : http://dcms.thailis.or.th/ dcms/
basic.php institute_code=54&option=show&bib=193&query=Bacteria%20&doc_type=0.
 100

(2543). Bacillus licheniformis KUB-

K0006 Bacillus pumilus KUB-K0082.
38.
. 522-528.
(2551). 2. .
(2542).
Morus alba L. 218
. (2542). .
. . 255
2555
: - .

. (2535). . -
284
. (2557).
127
(2548). Thin-layer Chromatography
Bacillus firmus Xanthomonas axonopodis pv.
glycines 43 .
313-320.
2555).

13.
2550
2550.

. (2552).
.
48. . 207-214.
 101

. (2553).
Xanthomonas oryzae pv. oryzae
.
47. 601-610.
. (2552).
Lactobacillus plantarum KMITNB 53.4
47. . 184-191.
, , , ,
(2549).

44. 795-810.
, , . (2550).

.
31 5. 264-265.
(2551).
.

. 2551 .
Erwinia carotovora subsp. carotovora .

. 120 .
2551).

, , ,
. (2550). Bacillus subtilis
. 30 2. 251-259.
. 2545 . .
. 173 .
. (2546). .
.
 102

, , . 2551 .
Bacillus spp. . .
. 39(3): 189-192.
2554). PR-1

120
2538 .

298
2540
. 92
(2545).
Xanthomonas oryzae pv. oryzae. .
. (2549).
.
1 . 46-47.
. (2550).
.
.
. (2552). .
. 77
. (2558). " .
.[ ]. :
http://www.prachachat.net/news_detail.php?newsid=
. (2546).
.
41. . 524-
531: 605
(2544).
.
 103

. (2529). .
.
(2542).
25
2547).
17-22.
, (2548). Xanthomonas
saxonopodis pv. dieffenbachiae Araceae
19: 47-56.
, . (2555).
Plant Growth Promoting Rhizobacteria (PGPR).
.
2536 Bacillus

31. 285-299: 683

(2534).

84
. (2551). .
LAB.TODAY 5. 38: 36-40.
(2554). Sphaceloma ampelinum
de Bary
. 55
(2550). Bacillus megaterium

. (2550). .

, . (2558).
Pseudomonas fluorescens .
4 (2): 155-164.
 104

2554 .
. 49.
148-155: 686
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Asaka, O. and Shoda, M. 1996 . Biocontrol of Rhizoctonia solani damping-off of tomato with
Bacillus subtilis RB 14. Applied and Environmental Microbiology. 62(11): 4081-4085.
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Bargabus, R.L., Zidack, N.K., Sherwood, J.E. and Jacobsen B.J. (2002). Characterization of
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 117

1.
1.1 Nutrient broth (NB)
Beef extract 3
Peptone 5
1,000
15 / 121
15-20
1.2 Nutrient Agar (NA)
Beef extract
Peptone
Agar 18
1,000

15
/ 121 15-20
 118

1 600
(Log of cell concentration (CFU.ml-1)) B. subtilis
CaSUT007

1 B. subtilis CaSUT007
600 nm B. subtilis
CaSUT007
0 0.021
12 0.135
24 0.321
48 0.363
72 0.394
 119

2 B. subtilis CaSUT007

2 B. subtilis
CaSUT007

cfu.g-1
0.1 1.9×105
0.2 3.8×105
0.3 5.7×105
0.4 7.6×105
0.5 9.5×105
 120

3 nutrient glucose
broth NGB) molasses diamonium phosphate and yeast extract MDY)
NGB MDY
/ /
Beef extract 3 16.2 25 0.175
Peptone 5 27.4 DAP 1 0.36
Glucose 10 0.3 Yeast extract 3 17.4
43.9 17.93

4 B. subtilis
CaSUT007

25 0.175
DAP 1 0.36
Yeast extract 3 17.4
9 0.4
CMC 1 0.55
18.88
100
 20 2533

2555
2555