71

CHAPTER 4

RESULTS

4.1. Isolation of fluorescent pseudomonad bacteria

Based on the heterotrophic plate count, the total population of culturable aerobic bacteria

and fluorescent bacteria associated with rice rhizosphere was 2.3×105 and 5.6×102 CFU/g

soil, respectively. A total of 443 fluorescent pseudomonad strains were isolated from the

rhizosphere soil.

4.2. Screening of antagonistic fluorescent pseudomonad bacteria

Out of 443 strains screened, strains FP10, PUP6 and PUW5 showed a broad-spectrum

antifungal activity towards phytopathogenic fungi used in the study and induced growth-

free inhibition zones (diameter) ranged from 6.3 to 32 mm towards phytopathogenic fungi

(Table 4).

Strains FP10, PUP6 and PUW5 also exhibited broad-spectrum activities against

bacterial pathogens of plant and human (Table 5). Strains induced growth-free inhibition

zones (diameter) ranged from 7.5 to 23 mm towards bacterial pathogens (Table 5).

72

Table 4. Broad-spectrum antagonistic activity against phytopathogenic fungi by strains FP10, PUP6 and PUW5

Test fungus

Disease

Host

Strains and inhibition zone diameter (mm ± SE)*

FP10 PUP6 PUW5

Sarocladium oryzae (SONS) Sheath rot Rice 14.6 ± 0.50d 26.0 ± 0.32b 19.3 ± 0.92a Rhizoctonia solani (RSR1) Sheath blight Rice 19.3 ± 0.17bc 19.0 ± 0.47d 14.0 ± 0.47bc Magnaporthe grisea (MGS) Blast Rice 20.6 ± 0.50b 32.0 ± 0.42a 19.3 ± 1.24a Fusarium oxysporum f.sp. cubense (FOC) Root wilt Banana 17.2 ± 0.56c 21.0 ± 0.19c 14.3 ± 1.20b Cylindrocladium floridanum (ATCC42971) Root necrosis Banana 16.3 ± 0.31c 20.0 ± 0.22cd 14.0 ± 0.67bc Cy. scoparium (ATCC 46300) Root necrosis Banana 22.4 ± 0.45ab 21.0 ± 0.61c 20.0 ± 0.33a Cy. spathiphylli (ATCC 44730) Root necrosis Banana 18.7 ± 0.38b 22.0 ± 0.28c 9.70 ± 0.47d Cy. spathiphylli (Gua5) Root necrosis Banana 25.4 ± 0.27a 32.0 ± 0.24a 9.70 ± 0.47d F. oxysporum f.sp. vasinfectum (FOV) Wilt Cotton 13.0 ± 0.57de 15.0 ± 0.36ef 13.0 ± 0.81c Colletotrichum falcatum (CFL) Red rot Sugarcane 9.70 ± 0.51f 16.0 ± 0.83e 12.6 ± 0.94c Macrophomina phaseolina (MPS) Charcoal rot Groundnut 12.6 ± 0.50e 14.0 ± 0.57f 11.3 ± 0.47cd Botrytis cinerea (BCTNAU) Blight Tobacco 10.3 ± 0.51ef 12.0 ± 0.63g 6.30 ± 0.47e C. capsici (CCL) Fruit rot Chili 12.3 ± 0.51ef 10.0 ± 0.59g 9.60 ± 0.47de Pestalotia theae (PTS) Leaf spot Tea 13.0 ± 0.57de 15.0 ± 0.17ef 17.0 ± 0.87ab C. gleosporoides (CGL) Anthracnose Mango 10.0 ± 0.57f 16.0 ± 0.34e 12.6 ± 1.69c

Means within the column followed by different letters are significantly different according to Duncan’s multiple range test p<0.05.

* Data represents the average of three replications. SE, standard error.

73

Table 5. Broad-spectrum antagonistic activity against plant and human pathogenic bacteria by strains FP10, PUP6 and PUW5

Test bacterium

Disease

Host

Strains and inhibition zone diameter (mm ± SE)*

FP10

PUP6

PUW5

Pseudomonas syringae pv. pisi 519 Bacterial blight Pea 8.20 ± 0.30a 0.0 7.50 ± 0.30a

P. syringae pv. glycenia NCPPB1783 Bacterial blight Glycene max 0.0 9.10 ± 0.40a 0.0

P. syringae pv. tomato NCPPB269 Bacterial speck Tomato 0.0 0.0 9.30 ± 0.20b

Staphylococcus aureus Scaled skin-syndrome Human 19.66 ± 1.15c 16.33 ± 0.47bc 23.00 ± 0.81d

Micrococcus luteus Skin infection Human 18.33 ± 0.33b 15.33 ± 0.47b 11.33 ± 0.47b

Bacillus sp. Skin infection Human 18.66 ± 0.47b 17.00 ± 0.81c 18.66 ± 0.47c

Candida albicans Candidiasis Human 21.66 ± 0.47d 18.33 ± 0.47c 19.33 ± 0.47c

Means within the column followed by different letters are significantly different according to Duncan’s multiple range test p<0.05.

* Data represents the average of three replications. SE, standard error.

74

4.3. Taxonomic characterization of antagonistic bacteria

4.3.1. Morphological, physiological and biochemical characterization

Morphologically strains FP10, PUP6 and PUW5 were smooth, circular and non-

mucoid, Gram-negative, motile and rod-shaped bacteria. All bacteria showed positive

reactions for fluorescence on King’s medium B (KB) agar. Strain FP10 tested positive

for cytochrome oxidase, arginine dihydrolase, gelatin hydrolysis, citrate utilization but

negative for levan formation and nitrate reduction. Strain FP10 utilized carbon

sources such as xylose, dextrose, galactose, arabinose, mannose, ribose, esculin,

malonate, sorbitol, mannitol and adonitol. This bacterium showed growth at 42oC but

did not grow at 4 oC (Table 6). Strain FP10 showed the optimum growth at 28oC.

Strain PUP6 tested positive for cytochrome oxidase, arginine dihydrolase,

gelatin hydrolysis and citrate utilization. This bacterium utilized L-arabinose, sorbitol

and mannitol as carbon sources but did not utilize other carbon sources used in this

study. Strain PUP6 showed growth at 42oC but did not grow at 4oC (Table 6). Strain

showed the optimum growth at 28oC. Strain PUW5 tested positive for the production

of arginine dihydrolase, cytochrome oxidase, nitrate reductase and citrate utilization

but negative for levan sucrase production and gelatin hydrolysis. Strain PUW5

showed optimum growth at 28oC but did not grow either at 4oC or 42oC. This

bacterium utilized lactose, xylose, fructose, dextrose, galactose, melibiose, L-

arabinose, mannose, ribose, α-methyl-D-mannoside, xylitol, esculin, D-arabinose and

mannitol as carbon sources sucrose but did not utilize glycerol, malonate, sorbose,

trehalose, sorbitol, adonitol and glucosamine (Table 6).

75

Table 6. Biochemical characterization of potent fluorescent pseudomonad strains

Trait

Strain

FP10

PUP6

PUW5

Fluorescence on KB + + + Oxidase + + + Arginine dihydrolase + + + Gelatin hydrolysis + + − Levan sucrase production − − − Citrate utilization + + + Nitrate reductase − − − Growth at 4°C − − − Growth at 42°C + + − Assimilation of - Lactose − − + Xylose + − + Fructose − − + Dextrose + − + Galactose + − + Melibiose − − + L-arabinose + + + Mannose + − + Glycerol − − − Ribose + − + α-Methyl-D-mannoside − − + Xylitol − − + Esculin + − + D-arabinose − − + Malonate + − − Sorbose − − − Trehalose − − − Sorbitol + + − Mannitol + + + Adonitol + − − Glucosamine − − −

+, positive reaction; −, negative reaction

76

4.3.2. Molecular characterization

4.3.2.1. Isolation, qualitative and quantitative analyses of genomic

DNA

Unsheared total genomic DNA was isolated from strain FP10, PUP6 and PUW5.

Genomic DNA showed the absorption ratio of 1.8 at 260 and 280 nm. The

concentration of the purified DNA was adjusted to 50 ng/ml in TE buffer.

4.3.2.2. Amplification and sequencing of 16S rRNA gene

Primers fD1 and rP2 amplified the 16S rRNA gene when the total genomic DNA of

FP10, PUP6 and PUW5 was used as templates in PCR. Agarose gel electrophoresis

and subsequent ethidium bromide staining of PCR products showed the 1500 bp

amplicons of 16S rRNA. The sequences (Chromas files, Applied Biosystems) were

manually checked for consistency of the results and used for molecular phylogenetic

analyses.

4.3.2.3. Molecular phylogenetic analyses

The 16S rRNA nucleotide sequence of strain FP10 showed 99% similarity towards P.

aeruginosa. Strain PUP6 showed 97% similarity towards P. aeruginosa and strain

PUW5 showed 98% similarity towards P. putida. On the basis of molecular

phylogenetic analysis of the 16S rRNA along with type strains, the taxonomic

affiliation of strains FP10 and PUP6 was confirmed as P. aeruginosa and strain

PUW5 as P. putida (Fig. 2A-C).

77

Figure 2A. Phylogenetic tree of strain FP10 based on the nucleotide sequence of

16S rRNA. The tree was constructed by neighbor-joining (NJ) method.

78

Figure 2B. Phylogenetic tree of strain PUP6 based on the nucleotide sequence of

16S rRNA. The tree was constructed by neighbor-joining (NJ) method.

79

Figure 2C. Phylogenetic tree of strain PUW5 based on the nucleotide sequence of

16S rRNA. The tree was constructed by neighbor-joining (NJ) method.

80

4.4. Determination of plant growth-promoting enzymes and

hormones

4.4.1. Production of siderophore

Strains FP10, PUP6 and PUW5 showed change in color of the medium from blue to

orange-red on CAS agar. The positive results of FeCl3 and hydroxamate tests

indicated the production of hydroxamate-type siderophore by strains FP10, PUP6 and

PUW5.

4.4.2. Production and estimation of phosphatase

Strains FP10, PUP6 and PUW5 produced phosphate solubilization on Pikovskaya’s

agar medium by inducing clear zones around the colonies. The solubilization of tri-

calcium phosphate by FP10, PUP6 and PUW5 after 10 days inoculation the soluble

phosphate was estimated to be 45.91 to 76.33 µg/ml (Table 6; Fig. 3A). The pH of

Pikovskaya’s liquid medium (pH 7.4) was reduced to pH 5.2 after 10 days inoculation

(Table 6; Fig. 3B).

4.4.3. Production and estimation of indole-3-acetic acid

Production of indole-3-acetic acid (IAA) was identified in strain FP10 and PUP6 by

the formation of a characteristic red halo on the filter paper immediately surrounding

the colonies. The IAA production was estimated during the stationary phase of

81

fermentation in Dworkin and Foster (DF) salts medium amended with 500 µg/ml of

tryptophan. Production of IAA by strains FP10 and PUP6 was estimated up to 19.1

µg/ml and 18.9 µg/ml, respectively (Table 7; Table 8).

4.4.4. Production of 1-aminocyclopropane carboxylate deaminase

Production of 1-aminocyclopropane carboxylate (ACC) deaminase was observed only

in strain PUW5 as shown by the growth on DF minimal salt medium amended with 3

mM ACC. Strains FP10 and PUP6 did not produce ACC deaminase (Table 7).

4.4.5. N-acyl homoserine lactone production

Strain FP10 produced N-acyl homoserine lactone (AHL) as observed by liberation of

the chromogen on the test medium (Table 7).

82

Table 6. Tricalcium phosphate solubilization by strains FP10, PUP6 and PUW5 in Pikovskaya’s medium*. Strain Days

1 3 5 7 10

P solubilized (µg/ml)**

pH P solubilzed

(µg/ml) pH P solubilzed

(µg/ml) pH P solubilzed

(µg/ml) pH P solubilzed

(µg/ml) pH

FP10 5.50 ± 0.09b 6.6 25.42 ± 0.70b 6.2 39.03 ± 0.10b 5.9 49.27 ± 0.52b 5.5 61.97 ± 0.40b 5.3 PUP6 5.08 ± 1.08a 6.6 19.92 ± 0.53a 6.1 30.31 ± 0.51a 5.9 38.37 ± 0.23a 5.7 45.91 ± 0.27a 5.5 PUW5 5.63 ± 0.69b 6.7 23.28 ± 0.05b 6.2 38.90 ± 0.08b 5.9 52.06 ± 0.10b 5.6 76.33 ± 0.70c 5.2

Means within the column followed by different letters are significantly different according to Duncan’s multiple range test p<0.05

SE, Standard error; *, pH of medium 7.4; **, Average of three replicates.

83

A

2 4 6 8 100

20

40

60

80

Ph

osph

ate

solu

biliz

ed (

µg/

ml)

Time (Days)

FP10 PUP6 PUW5

B

0 2 4 6 8 104.5

5.0

5.5

6.0

6.5

7.0

pH

Time (Days)

FP10 PUP6 PUW5

Figure 3. Determination of phosphate-solubilizing activity of strains FP10, PUP6

and PUW5. (A) Soluble phosphate was estimated from absorbance using the

calibration curve with KH2PO4 at 600 nm. The data obtained for strain PUP6 were

plotted as a function of time. (B) pH variation in Pikovskaya’s medium during growth

of strain PUP6.

84

Table 7. Plant growth-promoting traits of strains FP10, PUP6 and PUW5

Strain

Plant growth-promoting trait

Siderophore

IAA

Phosphatase

ACC deaminase

AHL

FP10 + + + − +

PUP6 + + + − −

PUW5 + − + + −

IAA, indole-3-acetic acid; ACC, 1-aminocyclopropane-carboxylate; AHL, N-acyl

homoserine lactone; +, positive reaction; −, negative reaction.

85

Table 8. Production of indole-3-acetic acid (IAA) in stationary phase culture of

strains FP10 and PUP6 in the presence of various concentrations of tryptophan

in Dworkin and Foster medium

Tryptophan (µg/ml)

IAA (µg/ml ±SE)*

FP10

PUP6

0 0.30 ± 0.1 0.10 ± 0.7

50 4.40 ± 0.4 4.80 ± 0.3

100 7.90 ± 0.8 8.00 ± 0.5

200 11.2 ± 0.6 10.3 ± 0.2

300 14.4 ± 0.3 13.6 ± 0.8

400 17.9 ± 0.2 17.3 ± 0.5

500 19.1 ± 0.9 18.9 ± 0.4

SE, Standard error; *, Average of three replicates.

86

4.5. Determination of potential for degradation of pesticides and

hydrocarbons

4.5.1. Utilization of pesticide/insecticide as sole carbon and energy

source

Strain FP10 was capable of growing on pesticide, lindane when provided as the sole

carbon and energy source. The typical growth pattern of strain FP10 is presented in

Fig. 4A. Strains PUP6 and PUW5 were capable of growing on insecticide, carbofuran.

The typical growth pattern of strains PUP6 and PUW5 is presented in Fig. 4B.

4.5.2. Biodegradation of pesticide, lindane

Percent biodegradation of lindane in the cultures was calculated on the basis of GC-

FID data. The percent biodegradation of lindane by strain FP10 was observed to be

99.22% (Fig. 5A).

4.5.3. Biodegradation of insecticide, carbofuran

Percent biodegradation of carbofuran in the cultures calculated on the basis of HPLC

with a UV detector (220 nm) analyses data. The percent biodegradation of carbofuran

by strains PUP6 and PUW5 was as observed to be 22.8% and 62.9%, respectively

(Fig. 5B).

87

A

0 2 4 6 8 10 120.0

0.2

0.4

0.6

Cel

l Den

sity

(OD

600)

Time (Days)

FP10

B

0 2 4 6 8 10 12

0.0

0.2

0.4

0.6

Cel

l Den

sity

(OD

600)

Time (Days)

PUP6 PUW5

Figure 4. Growth characteristics of strain FP10 in minimal medium amended

with lindane (A) and strains PUP6 and PUW5 in minimal medium amended with

carbofuran (B), when provided as the sole source of carbon and energy.

88

A

0 2 4 6 8 10 12

0

20

40

60

80

100

% re

mai

ning

lind

ane

Time (Days)

FP10

B

0 4 8 120

20

40

60

80

100

% r

emai

ning

car

bofu

ran

Time (Days)

PUP6 PUW5

Figure 5. Biodegradation of lindane by strain FP10 and carbofuran by strains

PUP6 and PUW5. The percent of remaining lindane (A) and carbofuran (B) in

minimal medium, when provided as the sole source of carbon and energy.

89

4.5.4. Utilization of n-alkane hydrocarbons and oils as sole carbon

and energy source

Strain PUP6 was capable of growing on small-chain (n-dodecane), medium-chain (n-

hexadecane and n-octadecane) and long-chain (n-octacosane) n-alkane members of

hydrocarbons when provided as the sole carbon and energy source, the typical growth

pattern is presented in Fig. 6A. Strain PUP6 was also capable of growing on complex

oils such as crude oil and lubricating oil. The typical growth pattern is presented in

Fig. 6B.

4.5.5. Biodegradation of n-alkane hydrocarbons and oils

The results of GC-FID analyses were used to calculate the percent biodegradation of

n-alkane members of hydrocarbons in the cultures, and the results are presented in

Fig. 7. On the basis of GC-FID analyses data, the percent biodegradation of

hydrocarbons by strain PUP6 was identified as: 97% n-dodecane, 21% n-hexadecane,

50% n-octadecane, and 53% n-octacosane (Fig. 7A). The percent biodegradation of

oils by strain PUP6 was observed to be 52% n-alkanes in crude oil and 74% n-alkanes

in lubrication oil (Fig. 7B).

90

A

B

Figure 6. Growth characteristics of strain PUP6 in minimal medium amended

with each n-alkane member of the hydrocarbons (A) and different oils (B), when

provided as the sole source of carbon and energy.

91

A

B

Figure 7. Biodegradation of hydrocarbons and oils by strain PUP6. Percent of

remaining hydrocarbons in minimal medium amended with each n-alkane (A) and

different oils (B), when provided as the sole source of carbon and energy.

92

4.6. Determination of fungal cell wall-degrading enzymes

4.6.1. Chitinase production

Strains FP10 and PUP6 produced extracellular chitinases as observed by the

formation of clear halos around the bacterial colonies on chitin agar plates (Table 9).

4.6.2. Protease production

Strains PUP6 and PUW5 produced extracellular protease as observed by inducing

clear halos around the bacterial colonies on skim milk agar plates (Table 9).

4.7. Determination of plant growth-inhibitory enzymes and

metabolites

4.7.1. Cellulase production

Strains FP10, PUP6 and PUW5 did not produce extracellular cellulase on congo red -

cellulose agar plates (Table 9).

4.7.2. Pectinase production

Strains FP10, PUP6 and PUW5 did not produce extracellular pectinase on pectin agar

plates (Table 9).

4.7.3. Hydrogen cyanide production

Strains FP10, PUP6 and PUW5 did not produce hydrogen cyanide (HCN) (Table 9).

93

Table 9. Production of fungal cell wall-degrading enzymes, plant growth inhibitory

enzymes and metabolites by strains FP10, PUP6 and PUW5

Strain

Fungal cell wall- degrading enzymes

Plant growth-inhibitory enzymes and metabolites

Chitinase

Protease

Cellulase

Pectinase

Hydrogen cyanide

FP10 + − − − −

PUP6 + + − − −

PUW5 − + − − −

94

4.8. Detection of antibiotic genes in fluorescent pseudomonads

When total genomic DNA was tested in PCR, primer pair, Phl2a and Phl2b amplified

the DNA fragment (745-bp) corresponding to that of phlD, which is a key gene,

involved in the biosynthesis of DAPG in the test strain FP10 and in the reference

strains P. fluorescens CHAO and P. fluorescens Pf-5, primer pair PhzHup and

PhzHlow amplified the DNA fragment (2000-bp) corresponding to that of PhzH,

which is a key gene in the biosynthesis of PCN in the test strain PUP6 and in the

reference strain P. aeruginosa PAO1. Whereas, all the primer pairs failed to amplify

the genes involved in the production of antibiotics such as DAPG, PCA, PCN, PLT

and PRN (Fig. 8; Table 10) in the test strain PUW5.

95

Figure 8. Detection of antibiotic genes by PCR in strains FP10, PUP6 and

PUW5. Lane M, 1 kb DNA ladder (Promega); Lane A, Positive control strain

(P. fluorescens Pf 2-79); Lane B, Positive control strain (P. aeruginosa PAO1);

Lane C, Positive control strain (P. fluorescens CHAO); Lane D, Positive control

strain (P. fluorescens Pf-5); Lane E, Positive control strain (P. fluorescens

CHAO); Lane 1, Strain FP10; Lane 2, Strain PUP6; Lane 3, Strain PUW5.

96

Table 10. Detection of antibiotic genes in strains FP10, PUP6 and PUW5 by PCR

Strain

Antibiotic gene (bp)

DAPG (745)

PCA (1100)

PCN (2000)

PRN (786)

PLT (779)

Reference strain

P. fluorescens Pf-5 + − − + +

P. fluorescens CHAO + − − + +

P. fluorescens 2-79 − + − − −

P. aeruginosa PAO1 − − + − −

Test strain

FP10 + − − − −

PUP6 – – + – –

PUW5 − − − − −

DAPG, 2,4-diacetylphloroglucinol; PCA, phenazine-1-carboxylic acid; PCN,

phenazine-1-carboxamide; PRN, pyrolnitrin; PLT, pyoluteorin ; +, positive reaction ;

−, negative reaction.

97

4.9. Production, extraction, purification and structural elucidation of

antifungal metabolites

4.9.1. Production and extraction of antifungal metabolites by FP10,

PUP6 and PUW5

Crude extracts by strains FP10, PUP6 and PUW5 showed dark-brown, brown and

orange-brown color, respectively. A total of 3.6 g, 3.4 g and 3.7 g of crude metabolite

was recovered from 10 L of fermentation cultures of strain FP10, PUP6 and PUW5,

respectively.

4.9.2. Purification and structural elucidation of antifungal metabolite

by strain FP10

4.9.2.1. Purification of antifungal metabolite by strain FP10

Crude extract by strain FP10 was purified through silica gel chromatography and

preparative HPLC. After purification, the crude extract yielded 13 mg of purified dark

brown metabolite. The active fractions were identified by in vitro antibiosis against

Cylindrocladium scoparium. TLC of purified metabolite showed the Rf value of 0.77

(Fig. 9A). The homogeneity of metabolite was further confirmed by analytical HPLC.

The metabolite was detected at a wavelength of 270 nm and its retention time was

10.77 min (Fig. 9B).

98

A

B

Figure 9. Thin layer chromatogram (A) high performance liquid chromatogram

(B) of antifingal metabolite produced by strain FP10.

99

4.9.2.2. Spectroscopic analyses of antifungal metabolite by strain

FP10

4.9.2.2.1. UV-Visible absorption spectroscopic analyses

The absorption spectrum of purified metabolite produced by strain FP10 showed

absorbance at 220 and 270 nm (Fig. 10).

4.9.2.2.2. Fourier transform infrared (FT-IR) spectroscopic analyses

FT-IR spectrum of purified metabolite produced by strain FP10 showed the functional

groups such as H-bonded OH, aromatic ring, C–H groups in C–CH3 compound, aryl

carbonyl compounds, C–OH in alcohols, ethers, acid esters (Fig. 11). Wave numbers

and corresponding groups present in the metabolite produced by FP10 are shown in

Table 11.

4.9.2.2.3. Liquid chromatography-Mass spectroscopic (LC-MS)

analyses of metabolite produced by FP10

The mass spectrum of purified metabolite produced by strain FP10 showed a

molecular ion peak of the metabolite at m/z 211.0 (M+H) (Fig. 12).

100

4.9.2.2.4. Structural elucidation of antifungal metabolite production

by FP10

On the basis of spectral data and the literature survey (Keel at al. 1990;

Shanahan et al. 1992) the metabolite has been characterized as 2,4-

diacetylphloroglucinol (DAPG) (Fig. 13). The minimum inhibitory concentration

(MIC) of DAPG towards M. phaseolina, S. aureus, M. luteus, Bacillus sp. S.

pyrogenes and C. albicans was 10 µg.

101

Figure 10. UV-visible abosorption spectrum of antifungal metabolite produced

by FP10.

102

Figure 11. FT-IR spectrum of antifungal metabolite produced by the strain FP10.

103

Table 11. Functional groups of infrared spectrum (FT-IR) of antifungal metabolite

produced by strain FP10

Wave numbers* (cm-1)

Functional groups

3600–3200 H-bonded OH

3420–3250 –OH group in alcohols and phenol

3300 Aromatic ring

2970–1850 C–H groups in C–CH3 compound

1700–1680 Aryl carbonyl compounds

1630–1430 Aromatic ring stretching

1600–1500 Aromatic ring

1200–1015 C–OH in alcohols

1100–130 Alcohols, ethers, acid esters

Functional groups were determined as described by Socrates (2001)

*An average of 40 scans in the frequency range 4000–500 cm-1

104

Figure 12. Mass spectrum of antifungal metabolite by strain FP10.

105

Figure 13. Structure of antifungal metabolite (2,4-diacetylphloroglucinol)

by strain FP10 based on UV-Visible, IR, NMR and MS data

106

4.9.3. Purification and structural elucidation of antifungal metabolite

by strain PUP6

4.9.3.1. Purification of antifungal metabolite by strain PUP6

Crude extract by strain PUP6 was purified through silica gel chromatography and

preparative HPLC. After purification, the crude extract yielded 10 mg of greenish-

yellow metabolite. The active fractions were identified by in vitro antibiosis against

Sarocladium oryzae. TLC of purified metabolite showed the Rf value of 0.48 (Fig.

14A). The homogeneity of metabolite was further confirmed by analytical HPLC. The

metabolite was detected at a wavelength of 254 nm and its retention time was 8.48

min (Fig. 14B).

107

A

B

Figure 14. Thin layer chromatogram (A) and high performance liquid

chromatogram (B) of antifungal metabolite by strain PUP6.

108

4.9.3.2. Spectroscopic analyses of antifungal metabolite by PUP6

4.9.3.2.1. Ultraviolet-visible absorption spectroscopic analyses

The absorption spectrum of purified metabolite produced by strain PUP6 showed

absorbance at 254 and 360 nm (Fig. 15).

4.9.3.2.2. Fourier transform infrared spectroscopic analyses

Fourier transform infrared (FT-IR) spectrum of purified metabolite produced by strain

PUP6 showed the functional groups such as NH2 streching, NH stretch in aromatic

amide, C-H stretch, several peaks due to aromatic ring, C=O stretch (amide bond),

aromatic ring stretching, C-N stretch (amide bond), C-N stretch, inplane ring bending,

ring deformation (Fig. 16). Wave numbers and corresponding groups present in

metabolite produced by PUP6 are shown in Table 12.

4.9.3.2.3. Liquid chromatography-Mass spectroscopic (LC-MS)

analyses

The mass spectrum of purified metabolite produced by strain PUP6 showed a

molecular ion peak of the metabolite at m/z 224.0 (M+H) (Fig. 17).

109

4.9.3.2.4. Nuclear magnetic resonance analyses of antifungal

metabolite produced by PUP6

The 1H- nuclear magnetic resonance (NMR) spectrum (500 MHz, CDCl3) of purified

metabolite produced by strain PUP6 showed signals due to the presence of seven

aromatic protons at δ 9.02 (1H, dd, J = 8.2, 2.1 Hz 2-H), 8.45 (1H, dd, J = 8.2, 2.1 Hz,

4-H), 8.32 (1H, ddd, J = 2.4, 5.2, 8.2 Hz, 9-H), 8.31 (1H, ddd J = 2.4, 5.2, 8.2 Hz, 6-

H), 7.97 (1H, dd, J = 8.2, 8.4 Hz, 3-H), 7.94 (1H, dd, J = 8.2, 2.2 Hz, 8-H), 7.92 (1H,

dd, J = 8.2, 2.2 Hz, 7-H). Furthermore, 1H NMR displayed two D2O exchangeable

signals at δ 10.8 (1H, brs NH) and 6.31 (1H, brs NH) (Fig. 18). The carboxamide

proton signals at δ 10.8 ppm confirmed the presence of carboxamide. The 13C NMR

spectra of the compound (120 MHz, CDCl3) showed peaks at δ 166, 143.5, 143.0,

141.5, 140.8, 134.3, 131.7, 131.0, 129.9, 129.1 and 128.8 ppm (Fig. 19). The proton

positions of the antibiotic were assigned using 1H-1H COSY, DQF-COSY, TOCSY,

HMBC, HSQC data (Fig. 20, 21, 22, 23, 24).

4.9.3.2.5. Structural elucidation of antifungal metabolite production

by PUP6

On the basis of spectral data and the literature survey (Chin-A-Woeng et al. 1998) the

metabolite has been characterized as phenazine-1-carboxamide (PCN) (Fig. 25). The

minimum inhibitory concentration of PCN towards S. oryzae, F. oxysporum f. sp.

cubense and S. pyrogenes was 60 µg.

110

Figure 15. UV-Visible abosorption spectrum of antifungal metabolite produced

by strain PUP6.

111

Figure 16. FT-IR spectrum of antifungal metabolite produced by strain PUP6.

112

Table 12. Functional groups of infrared spectrum (FT-IR) of antifungal metabolite

produced by strain PUP6

Wave numbers* (cm-1)

Functional groups

3540-3520 NH2 stretching

3520-3320 -NH stretch in aromatic amide

3100-3000 C-H stretch, several peaks due to aromatic ring

1680-1660 C=O stretch (Amide bond)

1630-1430 Aromatic ring stretching

1420-1400 C-N stretch (Amide bond)

1120-1030 C-N stretch

645-615 Inplane ring bending

580-420 Ring deformation

Functional groups were determined as described by Socrates (2001)

*An average of 40 scans in the frequency range 4000–500 cm-1

113

Figure 17. Mass spectrum of antifungal metabolite produced by strain PUP6.

114

Figure 18. 1H-NMR spectrum of antifungal metabolite produced by strain PUP6.

115

Figure 19. 13C-NMR spectrum of antifungal metabolite produced by strain PUP6.

116

Figure 20. 1H-1H-COSY NMR spectrum of antifungal metabolite produced by strain

PUP6.

117

Figure 21. DQF-COSY NMR spectrum of antifungal metabolite produced by strain

PUP6.

118

Figure 22. TOCSY-NMR spectrum of antifungal metabolite produced by strain

PUP6.

119

Figure 23. HMBC-NMR spectrum of antifungal metabolite produced by PUP6

120

Figure 24. HSQC-NMR spectrum of antifungal metabolite produced by PUP6

121

Table 13. Chemical shift assignments and coupling constant value of antifungal

metabolite produced by PUP6

Proton

Chemical shift (ppm)

Coupling constant J(H, H)/(Hz)

DQF-COSY 1H Cross peaks (ppm)

H-2 8.54 J(2, 3)=8.0, J(2, 4)=1.4 8.05

H-3 8.05 J(3, 2)=8.0, J(3, 4)=8.2 8.54, 8.98

H-4 8.98 J(4, 3)=8.2, J(4,2)=1.4 8.05

H-6 8.36 J(6, 7)=7.8, J(6, 8)=1.2 7.99

H-7 7.99 m 8.36, 8.05

H-8 8.05 m 7.99, 8.30

H-9 8.30 J(9, 8)=8.1, J(9,7)=1.0Hz 8.05

CONH2 10.8 s -

*s, singlet; m, multiplet

122

N

N

CONH2

2

345

5a 4a6

7

8

99a

1010a 1

Figure 25. Structure of antifungal metabolite (phenazine-1-carboxamide)

produced by strain PUP6 based on UV-Visible, FT-IR, NMR and mass

spectroscopic data

123

4.9.4. Purification and structural elucidation of antifungal metabolite

by strain PUW5

4.9.4.1. Purification of antifungal metabolite by strain PUW5

Crude extract by strain PUW5 was purified through silica gel chromatography and

preparative HPLC. After purification, the crude extract yielded 6 mg of purified

orange-brown metabolite. The active fractions were identified by in vitro antibiosis

against Sarocladium oryzae. TLC of purified metabolite showed the Rf value of 0.65

(Fig. 26A). The homogeneity of metabolite was further confirmed by analytical

HPLC. The metabolite was detected at a wavelength of 254 nm and its retention time

was 6.77 min (Fig. 26B).

124

A

B

Figure 26. Thin layer chromatogram (A) and high performance liquid

chromatogram (B) of antifungal metabolite by strain PUW5.

125

4.9.4.2. Spectroscopic analyses of antifungal metabolite by PUW5

4.9.4.2.1. UV-Visible absorption spectroscopic analyses

The absorption spectrum of purified metabolite produced by strain PUW5 showed

absorbance at 254, 360 and 430 nm (Fig. 27).

4.9.4.2.2. Fourier transform infrared (FT-IR) spectroscopic analyses

FT-IR spectrum of purified metabolite produced by strain PUW5 showed the

functional groups such as H-bonded OH, -CH antisym and sym stretching, CH3

attached to O or N, C=O in carboxylic acid group, COO- group in carboxylic acid,

aromatic ring stretching, C–OH in alcohols, O-C=O bending in carboxylic acids and

aromatic ring inplane deformation (Fig. 28). The transmittance corresponds to the

functional groups such as (Table 14).

4.9.4.2.3. Electrospray ionization mass spectroscopic (ESI-MS/MS)

analyses

ESI-MS spectrum of purified compound produced by PUW5 showed a molecular ion

peak of the metabolite at m/z 241.1 (M+H) and m/z 263.0 (M+Na+H). The second

dimension mass spectrum (MS/MS) of molecular ion peak m/z 241.1 (M+H), resluted

fragmentation pattern consisting molecular ion peaks at m/z 225.0, m/z 208.1 and m/z

180.1 (Fig. 29).

126

4.9.4.2.4. NMR analyses of antifungal metabolite produced by PUW5

The 1H NMR spectrum (500 MHz, CDCl3) of purified antibiotic isolated from strain

PUW5, showed signals due to the presence of seven aromatic protons at δ 9.00, 8.56,

8.37, 8.31, 8.07, 8.05, 7.99 ppm and carboxylic acid and methyl proton at δ 15.6 and

1.57, respectively (Fig. 30). The 13C NMR spectra of the compound (125.7 MHz,

CDCl3) showed peaks at δ 165.93, 147.11, 144.13, 143.49, 140.54, 137.46, 135.17,

133.24, 131.76, 130.29, 130.15, 127.99, 125.13, and 29.71 ppm (Fig. 31). The proton

positions of the antibiotic were assigned using 1H-1H COSY, DQFCOSY, TOCSY

and HMQC data (Fig. 32, 33, 34, 35; Table 15).

4.9.4.2.5. Structural elucidation of antifungal metabolite production

by PUW5

On the basis of NMR, mass spectroscopic data and literature survey the metabolite

has been identified as 5-methyl phenazine-1-carboxylic acid betaine (MPCB) (Fig.

36). The minimum inhibitory concentration (MIC) of MPCB towards S. oryzae, M.

grisea and S. pyrogenes was 60 µg.

127

Figure 27. UV-Visible abosorption spectrum of antifungal metabolite produced

by strain PUW5.

128

Figure 28. FT-IR spectrum of antifungal metabolite produced by strain PUW5.

129

Table 14. Functional groups of infrared spectrum (FT-IR) of antifungal

metabolite produced by strain PUW5

Wave numbers* (cm-1)

Functional groups

3600–3200 H-bonded OH

2990-2880 -CH antisym and sym stretching

2850-2700 CH3 attached to O or N

1710-1690 C=O in carboxylic acid group

1400-1310 COO- group in carboxylic acid

1630–1430 Aromatic ring stretching

1200-1015 C–OH in alcohols

700-590 O-C=O bending in carboxylic acids

545-520 Aromatic ring inplane deformation

Functional groups were determined as described by Socrates (2001)

*An average of 40 scans in the frequency range 4000–500 cm-1

130

Fig. 29. ESI-MS and ESI-MS/MS spectrum (inset) of the antifungal metabolite

produced by strain PUW5.

131

Fig. 30. 1H-NMR spectrum of antifungal metabolite produced by strain PUW5.

132

Fig. 31. 13C-NMR spectrum of antifungal metabolite produced by strain PUW5.

133

Fig. 32. 1H, 1H- COSY NMR spectrum of antifungal metabolite produced by strain

PUW5.

134

Fig. 33. DQF-COSY NMR spectrum of antifungal metabolite produced by strain

PUW5.

135

Fig. 34. TOCSY-NMR spectrum of antifungal metabolite produced by PUW5.

136

Fig. 35. HSQC-NMR spectrum of antifungal metabolite produced by strain

PUW5.

137

Table 15. Chemical shift assignments and coupling constant value of antifungal

metabolite produced by strain PUW5

Proton

Chemical shift (ppm)

Coupling constant J(H, H)/(Hz)

DQF-COSY 1H Cross peaks (ppm)

H-2 8.56 J(2, 3)=5.5, J(2, 4)=9.0 8.05

H-3 8.05 J(3, 2)=7.5, J(3, 4)=8.5 8.56, 8.99

H-4 8.99 J(4, 3)=6.5, J(4,2)=1.0 8.05

H-6 8.35 J(6, 7)=7.0, J(6, 8)=1.0 7.98

H-7 7.98 m 8.35, 8.05

H-8 8.05 m 7.98, 8.30

H-9 8.30 J(9, 8)=8.0, J(9,7)=1.0Hz 8.05

COOH 15.6 s -

CH3 1.5 brs -

*s, singlet; m, multiplet; brs, broad singlet

138

Fig. 36. Structure of antifungal metabolite (5-methyl phenazine-1-carboxylic acid

betaine) produced by strain PUW5 based on FT-IR, NMR and ESI-MS/MS data.