4.1 isolation and screening of bacillus sp -...

Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............

Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 92

4.1 Isolation and screening of Bacillus sp.

A total of 15 chilli rhizosphere soils were serially diluted and subjected to heat

treatment. Isolation and screening of such heat treated samples was conducted on colloidal

chitin amended plates. Out of 15 chilli rhizosphere soil samples plated on minimal salts agar

amended with colloidal chitin, 9 chitinolytic colonies (isolates numbered 1 - 9) were

obtained from the soil samples numbered 2, 4, 6, 7, 9, 11 and 13 based on the clearance

zones produced.

These chitinolytic isolates were subjected to dual plate assay with nine different

chilli fungal pathogens (Table 4.1). Isolates 3, 4, 5 and 6 expressed antagonism against

Alternaria brassicicola (22%), Fusarium oxysporum (15%), Verticillium theobromae (33%)

and Rhizoctonia solani (12%), respectively. The isolate 1 was found to be antagonistic to six

fungal pathogens with an inhibition percentage ranging from 22-39%. However, isolate 2

expressed antibiosis against all the nine fungal pathogens with an inhibition percentage

ranging from 40-62%. The isolate showed antagonism against Alternaria alternata (45%),

Alternaria brassicae (57%), Alternaria brassicicola (53%), Colletotrichum gloeosporioides

(57%), Phytophthora capsici (62%), Rhizoctonia solani (56%), Fusarium solani (41%),

Fusarium oxysporum (40%) and Verticillium theobromae (52%), (Plate 4.1) (Table 4.2).

Further the antagonistic activity of the cell free culture filtrate against all the

pathogens was evaluated in comparison to the respective pathogen controls without the

culture filtrate. The results of the percentage inhibition in pathogen dry weight are presented

in the Table 4.2. In the presence of the culture filtrate, a percentage inhibition ranging from

58 to 84% was observed by evaluation of the reduction in biomass of the fungal pathogens.

The culture filtrate exhibited maximum percentage dry weight reduction of the

C.gloeosporioides and R.solani mycelium, in comparison to the control (without culture

filtrate) and other pathogen mycelia.



The isolate numbered 2 expressed a broad spectrum of antagonistic activity against

all the nine fungal pathogens, evident from dual plate assay (40-62%) and reduction in dry

weight in the presence of the culture filtrate (58-84%) and was chosen for further studies.

This pattern of antagonism was in agreement to Kamil et al. (2007) who reported a

broad spectrum of inhibition of fungal phytopathogens by chitinolytic Bacillus sp. and

Thakaew and Niamsup (2013) who reported inhibition (89.6%) of aflatoxigenic fungus

Aspergillus flavus by Bacillus subtilis BCC 6327. It has been reported that antifungal

mechanism of antagonists has been attributed to the action of hydrolytic enzymes such as

chitinase, β-1, 3-glucanase, chitosanase, and protease (Wang et al., 1999; Wang et al., 2002;

Chang et al., 2007). The results of the present study are also in concordance with Thakaew

and Niamsup (2012) who reported the reduction in aflatoxigenic fungi mycelial production

with inhibition percentages of 92.1, 89.6 and 90.1% from cell free supernatants of B.subtilis

at 12, 24 and 36 h, respectively.



Table 4.1. Dual plate assay showing % inhibition of the chitinolytic Bacillus sp.

PHYTOPATHOGENS

% INHIBITION OF THE CHITINOLYTIC

ISOLATES

1 2 3 4 5 6 7 8 9

Colletotrichum

gloeosporiodes(OGC1)

39

57

0

0

0

0

0

0

0

Alternaria brassicae

(OCA1)

32

57

0

0

0

0

0

0

0

Alternaria brassicicola

(OCA3)

25

53

22

0

0

0

0

0

0

Alternaria alternata

(OTA36)

36

45

0

0

0

0

0

0

0

Phytophthora capsici

(98-01)

22

62

0

0

0

0

0

0

0

Verticillium theobromae

(MTCC 2066)

22

52

0

0

33

0

0

0

0

Fusarium solani

(MTCC 1756)

0

41

0

0

0

0

0

0

0

Fusarium oxysporum

(MTCC 1755)

0

40

0

15

0

0

0

0

0

Rhizoctonia solani

(MTCC 4633)

0

56

0

0

0

12

0

0

0



Table 4.2. Antagonistic activity of Bacillus sp against different phytopathogens.

Results are the mean of 3 replicates ± SD. In columns, values with the same letters are not

significantly different (P < 0.05 Duncan test)

SL

NO

Pathogens

Bacillus sp. %

Inhibition

(Dual plate assay)

% Inhibition

(Dry Weight)

1.

Colletotrichum gloeosporiodes(OGC1)

57±0.901d

84±6.02d

2.

Alternaria brassicae (OCA1)

57±2.685a

64±7.211a,b

3.

Alternaria brassicicola (OCA3)

53±1.443a

58±5.8a

4.

Alternaria alternata (OTA36)

45±1.01c

69±5.85a,b,c

5.

Phytophthora capsici (98-01)

62±0.935c

78±8.5c,d

6.

Verticillium theobromae

(MTCC 2066)

52±2.049c

60±5a,b

7.

Fusarium solani

(MTCC 1756)

41±1.527b

66±6.02a,b

8.

Fusarium oxysporum

(MTCC 1755)

40±1.607e

70±5b,c

9.

Rhizoctonia solani

(MTCC 4633)

56±5.204f

80±2.51c,d



Plate 4.1. Dual Plate assay of Bacillus sp against different fungal phytopathogens –

A. Alternaria brassisicola; B. Alternaria alternata; C. Alternaria brassicae;

D. Colletotrichum gloeosporioides; E. Rhizoctonia solani; F.Verticillium theobromae;

G. Fusarium oxysporum; H. Fusarium solani and I. Phytophthora capsici on PDA.



4.2 Morphological and phenotypic characterization of Bacillus sp.

Chitinolytic isolates of Bacillus genus were characterized according to the Bergey’s

Manual of determinative Bacteriology (Holt, 1994). Microscopic studies revealed that the

cells were rod-like, motile, gram-positive, catalase positive, and spore-forming under

aerobic conditions. The isolate was checked for other biochemical characteristics (Table

4.3) and was tentatively identified as Bacillus sp. The isolate was positive for the following

tests: Catalase, Nitrate, Citrate, Voges Proskauer, growth in 6.5% NaCl plate incubated at

55°C and fermentation of glucose and mannitol. The isolate was negative for the following

tests: Oxidase, parasporal crystal formation and fermentation of sucrose and lactose.

The 16S rRNA gene analysis identified the isolate as belonging to the genus

Bacillus. 16S rDNA sequence analysis showed that this Bacillus strain had 100% similarity

with B.subtilis (Fig. 4.1), and was designated as B. subtilis and the Gene-Bank accession no.

for the nucleotide sequence was JN032305.1.



Table 4.3. Phenotypic characterisation of bacterial isolate

Test Results

Colony morphology Smooth

Gram stain Gram positive

Cell shape Rod

Spore formation +

Motility +

Parasporal crystal -

Catalase +

Oxidase -

Nitrate reduction +

Citrate utilization +

Voges Proskauer +

Growth on 6.5% NaCl at

55°C +

Carbohydrate utilization

Glucose +

Sucrose -

Lactose -

Mannitol +



Fig. 4.1. Phylogenetic tree of the Bacillus subtilis based on the 16S rDNA sequences.



4.2.1 Intrinsic antibiotic resistance

Antibiotics resistance pattern study of B.subtilis revealed the isolate to be highly

susceptible to Streptomycin (10µg), moderately sensitive to tetracycline (30µg) and

Kanamycin (10µg) and resistant to Nalidixic acid (50µg) (Plate 4.2). The susceptibility of

bacilli to different antibiotics has been studied previously and it has been demonstrated that

in principle it should be possible to identify species on the basis of the results of

susceptibility tests (Coonrod et al., 1971). A large number of Bacillus strains assigned to

different species were tested to determine their susceptibilities to antibiotics. Considerable

interspecific differences in susceptibility were observed with chloramphenicol, ampicillin,

and tetracycline (Reva et al., 1995).

4.2.2 Compatibility study of B.subtilis with fungicides

B.subtilis was compatible with Carbendazim (Bavistin) and Chorothalonil (Kavach),

both standard fungicides used to control root rot and anthracnose in Solanaceae crops (Plate

4.3). This compatibility affords an opportunity for the integration of chemical with

biological agents. Since fungicides may have deleterious effects on the pathogen as well as

the antagonist, an understanding of the effect of fungicides on the pathogen and the

antagonist, would provide information on the selection of selective fungicides and fungicide

resistant antagonists. The idea of combining biocontrol agents (BCA) with fungicides is for

the development or establishment of desired microbes in the rhizosphere (Papavizas and

Lewis, 1981). Further, the antagonism of BCA was influenced by the addition of fungicides

(Kay and Stewart, 1994; Naar and Kecskes, 1998). The results are in concordance with

many authors who reported the compatibility of fungicides with biocontrol agents in various

crops (Utkhede and Koch, 2002; Senthilvel et al., 2004; Anand et al., 2007).


Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. 1

Plate 4.2. Intrinsic Antibiotic resistance pattern of Bacillus subtilis to Nalidixic acid

and Kanamycin (A) ;Tetracycline and Streptomycin (B)

Plate 4.3. Fungicide tolerance of Bacillus subtilis with standard fungicides

Carbendazim (Bavistin) and Chlorothalonil (Kavach).



4.3 Characterisation of Bacillus subtilis for PGPR activities and hydrolytic enzymes

The isolate under study, was evaluated for direct and indirect plant growth

promotion traits. The isolate exhibited the ability to produce mycolytic enzymes checked for

namely, chitinase, β 1,3 and β 1,4 glucanase, protease and lipase. Evaluation of PGPR

activities indicated absence of traits such as IAA, siderophore and HCN production. The

isolate was negative for Phytase production and phosphate solubilisation also. The isolate

was positive for indirect growth promotion abilities in the form of production of mycolytic

enzymes but negative for direct growth promotional traits such as IAA, siderophore, HCN,

Phytase and PO4 solubilization. The chitinolytic isolate additionally produced hydrolytic

enzymes like β-1, 3 and β-1, 4 glucanase, as evidenced from the clearance zones on yeast

glucan plates as well as CMC plates, respectively. The isolate was positive for protease and

lipase and the ability was established due to the clearance zone produced on milk agar plate

and fluorescence detected on olive oil amended plate with rhodamine-B respectively (Table

4.4).

Bacteria commonly produce cell wall-degrading enzymes and secondary metabolites

to hinder the growth of other micro-organisms (Shoda, 2000). Mycolytic enzymes produced

by antagonistic microorganisms are very important in biocontrol technology. There are

many reports on production of lytic enzymes by microorganisms (Baharum et al., 2003;

Huang et al., 2004; Gohel et al., 2004). In recent years microbial lytic enzymes such as

chitinase, and β-1,3-glucanase have been exploited for the management of plant diseases. β-

glucanases, produced by several fungi and bacteria are one of the most potent enzymes for

degrading fungal cell walls (Bodenmann et al., 1985; Stahmann et al., 1993; Lorito et al.,

1994. Aktuganov et al., 2007) have reported that among the hydrolases secreted by Bacillus

sp. 739, β-1, 3-glucanases and chitinases most actively degraded the disintegrated cell-wall



material of B. sorokiniana. Cellulases are a class of enzymes which help in antagonism.

These enzymes used by bacterial isolates digest the cellulose present in the cell walls of

pathogens (Brantlee et al., 2011). From the studies reported by Helistö and collaborators

(2001) on Bacillus sp. X-b, a biocontrol agent against plant pathogenic fungi, secretes a

complex of enzymes composed of chitinase, laminarinase, lipase and protease which play a

major role in antagonism against fungal pathogens. This is in agreement to the present

study.

Many species and specific strains of bacteria colonising rhizosphere have been

shown to possess plant growth promoting traits and hence they are collectively designated

as plant growth promoting rhizobacteria (PGPR) (Gaskins et al., 1985). PGPR enhance

plant productivity by several mechanisms. These beneficial effects of PGPR can be either

direct or indirect. Direct promotion of growth by PGPR occurs when the rhizobacteria

produce metabolites that promote plant growth such as auxins (Asghar et al., 2002),

cytokinins (Arkhipova et al., 2005) and gibberellins (Gutierrez- Manero et al., 2001; Joo et

al., 2004) as well as through the solubilization of phosphate minerals (Freitas et al., 1997).

PGPR beneficial effects have been exploited in many areas including biofertilizers,

microbial rhizoremediation and biopesticides. PGPR beneficial effects have been exploited

in many areas including biofertilizers, microbial rhizoremediation and biopesticides

(Adesemoye et al., 2008). Such direct growth promotion is not observed in the present

study.



Table 4.4. Mycolytic Enzyme profile and PGPR traits of the isolate

TEST

ISOLATE

CHITINASE

+

β-1,4 GLUCANASE

+

β-1,3 GLUCANASE

+

PROTEASE

+

LIPASE

+

IAA

-

HCN

-

SIDEROPHORE

-

PO4 SOLUBILIZATION

-

PHYTASE PRODUCTION

-



Plate 4.4. Mycolytic enzyme profile of the isolate.



4.3.1 Antifungal assay of the cell free supernatant by agar diffusion method (diffusible

metabolite)

The radial growth of the pathogen, C.gloeosporioides was measured in the control

plate and the plate containing the day wise culture filtrate. The results (Plate 4.5) showed

inhibition of colony diameter in the test plates in comparison to the control plate which did

not contain the extract. The crude culture filtrate showed 45% and 50% inhibition in the

plates of day 1, day 2 respectively, while 100% inhibition was achieved with the cell free

extracts of day 3-5. The results indicated that fungal growth suppression resulted from the

presence of extracellular antifungal metabolites in culture filtrates. Similar results have been

reported by Islam et al. (2012) in B.subtilis and El-Abyad et al. (1996) in Streptomyces sp.

Devkota et al. (2011) have showed maximum inhibition of F.oxysporum upto 53.29% with

the fourth day culture filtrate incubated at 37°C



Plate 4.5. Antifungal assay of the day wise cell free culture supernatant against the

pathogen C.gloeosporioides on MRBA plates. A. Test plates B. Control



4.4 Optimisation of Growth Conditions For Maximum Production of Enzymes

4.4.1 Media optimisation

Culture medium is a key factor for the growth as well as metabolite production by

the microorganisms. In the present study, all the three media (NB, LB and YNB) under

evaluation were able to support the production of all the three mycolytic enzymes from day

one (Fig. 4.2). In LB broth and YNB the chitinase activity peaked on day one followed by a

decline in activity on the consecutive days but the activity of the glucanases (β 1,3 and β

1,4) was maximum on the third day. As evident from the graph, NB amended with colloidal

chitin supported maximum activity of all three hydrolytic enzymes with peak activity on day

3 (10 U/ml for chitinase, 8 U/ml for β 1, 3 glucanase and 13 U/ml for β 1,4 glucanase).

Hence Nutrient broth was chosen as the best basal medium for optimization study. This is in

contrast to the work by Kavi Karunya et al. (2011) reporting Luria Bertaini broth for highest

chitinase production of 28 units/ml as compared to Nutrient broth, and Shanmugaiah et al.

(2008) who have reported in their work with B.laterosporous MML2270, Yeast Nitrogen

base medium as the most suitable medium for high chitinase production of 19.7 U/ml as

compared to 15.0 and 14.3 U/ml, respectively in Luria Bertaini and Nutrient broth.



Fig. 4.2: Medium optimization for the production of Chitinase (a), β 1,3 Glucanase (b)

and β 1,4 Glucanase (c).



4.4.2 Induction with pure substrates and fungal mycelium

The level of secretion of mycolytic enzymes in the monoculture of BCA depends

mainly on the content of specific substrates in the nutrient medium. The induction profile of

the three mycolytic enzymes with pure substrates such as colloidal chitin, yeast cell glucan,

carboxy methyl cellulose (CMC) supplemented in NB was studied. The addition of CMC to

NB proved to be the best for all the three enzymes, giving activity of 14 U/mL for chitinase,

17 U/mL for β 1,3 glucanase and 22 U/mL for β 1,4 glucanase (Figure 4.3). Such a

combined induction of all the mycolytic enzymes with the supplementation of a single

substrate would be beneficial for the optimal activity of the BCA. This is in accordance

with the fact that most of the mycolytic systems reported in the literature are inducible

(Gohel et al., 2004; Monreal and Reese, 1969; Vyas and Deshpande, 1989). Further the

evaluation for the optimal percentage of CMC suggested 1% (w/v) (Figure 4.4) to be best

suited for the optimal production of all the three enzymes. This was in contrary to, colloidal

chitin at 0.3% (w/v) reportedly supporting highest production of chitinase and

β-1,3 glucanase (Kavikarunya et al., 2012; Leelasuphakul et al., 2006). Chitosan also has

been reported to synergistically induce and enhance the production of lytic enzymes such as

chitinase, cellulase and β-glucanases (Shadia and Abdel-Aziz, 2013). The alkaline cellulase

from Bacillus subtilis AS3 showed multisubstrate specificity showing activity with CMC,

laminarin, hydroxyethylcellulose, and steam exploded bagasse and significantly higher

activity with lichenan and barley β-glucan (Deepmoni et al., 2011). In similar lines, there is

a possibility that the mycolytic enzyme of B.subtilis also may be a single enzyme with

multisubstrate activity. This needs to be further explored.



Fig. 4.3: Induction of mycolytic enzymes with pure substrates: Chitinase (a), β 1,3

Glucanase (b) and β 1,4 Glucanase (c)



Fig. 4.4. Optimization of CMC concentration (%, w/v) for maximum mycolytic

enzyme activity.



The induction profile of the B. subtilis was checked with autoclaved

C. gloeosporioides and R. solani mycelium used as the carbon source in the medium and

was compared with the control. Data represented in Figure 4.5 showed that the dead mycelia

of C. gloeosporioides and R. solani were able to induce the production of all the three

hydrolytic enzymes by B. subtilis. The production of the lytic enzymes was followed by

lysis of the mycelia, suggesting the possible role of these enzymes in antibiosis of the

pathogens. On dead mycelium of C. gloeosporioides, greater quantity of chitinase was

produced and moderately high quantities of β-1, 3-glucanase and β -1, 4-glucanase were

produced by the antagonistic strain. But on dead mycelium of R. solani, chitinase was

moderately produced but greater quantity of β-1, 3-glucanase, and β -1, 4-glucanase was

produced. This was supportive of the difference in cell wall composition of the two

pathogens, the former being an ascomycete and the latter being a basidiomycete.

Several researchers (Fayad et al., 2001; Hayat et al., 2010) report the production of

extracellular enzymes by various microorganisms as hydrolytic enzymes which attack the

structural components of cell walls of most fungi. Diby et al. (2005) reported hyphal wall

components of Phytophthora capsici, both as fresh and dried, as the best substrate for the

production of chitinase and β 1,3 glucanase by Pseudomonas fluorescens and Trichoderma

sp. in comparison to other substrates. Significant activities of β-1,3-glucanase and chitinase

were produced byT. harzianum in culture media amended with dried or fresh mycelium of

S. Rolfsii (El-Katatny et al., 2000). Similarly Soledad et al. (1998) extensively studied the β

1,3 glucanase of Trichoderma harzianum and reported that laminarin and dried mycelia

induced the production of glucanases, the highest, in comparison to other substrates. Eman

Zakaria Gomaa (2012) reported a two fold enhancement of chitinase production by B.

thuringiensis and B. licheniformis in colloidal chitin medium amended with dried fungal

mats. The results are also in concordance with Thakaew and Niamsup (2013) who have



reported enhancement of production of protease, chitinase and β-1,3-glucanase and the

released sugars (total reducing sugar, glucose and N-acetylglucosamine) by B.subtilis in the

presence of dried fungal mycelia of aflatoxigenic fungi. The present study is in agreement

to the above mentioned reports which indicate that when the pathogen mycelium is provided

as a substrate for growth, different levels of the hydrolytic enzymes are induced

proportionate to the content of the respective substrate in the cell wall.



Fig. 4.5: Induction of hydrolytic enzymes with autoclaved mycelium of

C.gloeosporioides (a) and R.solani (b)



4.4.3 Hydrolytic activity of B. subtilis culture filtrate

The results of the hydrolytic activity of the mycolytic enzymes produced by

B.subtilis are represented in Figure 4.6. The results were obtained and evaluated in the

presence of the pathogen mycelium as substrate for the assay. The crude culture filtrate

which was used for the enzyme assay showed appreciable hydrolytic activity with both

C.gloeosporioides and R.solani mycelia as substrate. The results indicated higher activity

with R.solani mycelium and moderate activity with C.gloeosporioides mycelium. These

observations were supportive to the results obtained in mycelium induction of hydrolytic

enzymes.The objective of the present study was to use B.subtilis as a biocontrol agent

against fungal pathogens. Hence it is necessary to assess the hydrolytic activity of the

culture filtrate by using the fungal mycelium as the substrate for assay rather than any other

pure substrate. The results were in agreement with El-Katatny et al. (2000) who reported

crude culture filtrates of T. harzianum to possess hydrolytic activity on dried or fresh

mycelium of the phytopathogenic fungus S. rolfsii. B. subtilis culture filtrates, possessing

protease, chitinase and β-1,3-glucanase. These were capable of hydrolyzing dried mycelia of

the isolated aflatoxigenic fungi from bird chilli powder (Thakaew and Niamsup, 2013).



Fig. 4.6: Hydrolytic activity of B.subtilis on autoclaved fungal mycelium.



4.4.4 Microscopy

The fungal mycelium of C. gloeosporioides and R. solani grown with B. subtilis

culture showed damage, swelling and distortions as compared to the control which did not

show these abnormal features (Fig. 4.7). This clearly indicates the mycolytic activity of the

B. subtilis. A similar observation has been made in the antagonism of Arthrobacter sp. to

Fusarium sp. (Barrows-Broaddus and Kerr 1981). Similarly, Podile and Prakash (1996)

reported the lysis and dissolution of fungal mycelium of Aspergillus niger by B. subtilis

AF1 strain. Tendulkar et al. (2007) conducted microscopic analysis of the effect of Bacillus

licheniformis BC98 on M. grisea, revealing bulbous hyphae showing patchy and vacuolated

cytoplasm when observed under the electron microscope. Similarly, light microscopic

observations of the bulbous and swollen germinating spores and hyphal tips revealed

shrunken, granulated and vesicular cytoplasm as compared with the hyaline, healthy

cytoplasm of control untreated hyphae.



Fig. 4.7: Light microscopic observations of mycelium inhibited by B.subtilis. Mycelium

of R.solani (1a) and C. gloeosporioides (2a ) grown on PDA; present in the inhibition

zone when grown along with B.subtilis on PDA (1b and 2b)



4.4.5 Effect of media components on mycolytic enzyme production

4.4.5.1 Carbon sources

In this study, various carbon sources were evaluated for their effect on mycolytic

enzyme production by B.subtilis. The supplementation of the medium with organic carbon

such as CMC, colloidal chitin, Yeast Cell Wall (YCW), Avicel had a positive influence on

the production of all the three lytic enzymes in varied levels, in comparison to the control

(NB). In the present study, addition of simple sugars like glucose, lactose, maltose and

sucrose in the medium as sole carbon source repressed the activity of chitinase and β-1,4

glucanase. However, addition of glucose and maltose had an enhancing effect on production

of β 1,3 glucanase. Glucose supplemented with chitin or CMC was identified as the best

carbon source among all the carbon sources checked (Fig. 4.8) and particularly in

comparison to the control. A significant increase (P≤0.05) (Table 4.5) in chitinase by 4.15

folds; glucanase by 6.28 folds and cellulase by 1.95 folds was obtained with the

supplementation of glucose and CMC in NB media.

Similar observation has been reported by Andronopoulou and Vorgias (2004) for

chitinase production by Thermococcus chitonophagus. Medium composition is one of the

main factors that enhance chitinase and cellulase production by microorganisms (Al-

Ahmadi et al., 2008; Akhir et al., 2009; Faramarzi et al., 2009; Immanuel et al., 2006). El-

Katany et al. (2000) reported that 0.5% glucose addition to chitin substrate repressed

chitinase production by T.harzianum. Ghanem (1992) found that addition of glucose was of

repressive action on chitinase production by Bacillus amyloliqefaciens. Lopes et al. (2008)

found that chitinase production by Moniliophora perniciosa was repressed by the addition

of glucose.



Fig. 4.8: Effect of different Carbon sources on mycolytic enzyme production by

B.subtilis.

Table 4.5. ANOVA for Carbon effect on mycolytic enzymes production by B.subtilis

Chitinase Glucanase Cellulase

Mean square 7.10 0.843 73.10 0.48 56.12 0.88

F-value 8.422 152.29 63.17

P(5%) 2.45 (S)*

2.36(S)*

2.45(S)*



Ahamed and Vermette (2008) and Domingues et al. (2000) have reported that CMC

shows inducing effect on cellulase production. It has been reported that biosynthesis of

cellulases in Trichoderma reesei was very high in medium with CMC as carbon source

(Ghanem, 1992; Gohel et al., 2006). These results are in agreement with those of Narasimha

et al. (2006) and Niranjane et al. (2007) who found that CMC was the best carbon source

followed by cellulose for cellulase production.

A higher production of cellulase when CMC served as substrate due in part to

induction of the enzyme, since cellulose is known to be a universal inducer of cellulase

synthesis. Paul and Varma (1993) had reported the induction of endocellulase by CMC.

Medium containing glucose as the carbon source presented the minimum cellulase activity.

Viruthagiri and Muthuvelayudham (2006) obtained similar results which showed that the

cellulase activity was less when glucose was used as carbon source because of inhibition.

NB with CMC supported appreciable enzyme levels, hence was media of choice for

further experiments. In contrast to earlier studies wherein medium optimization have

focused on any single enzyme with their respective substrates, the present study reports

concerted production of three different mycolytic enzymes using CMC as substrate.

4.4.5.2 Nitrogen sources

Among various organic and inorganic sources tested for mycolytic enzyme

production by Bacillus subtilis, CSL, KNO3, casein and yeast extract supplementation in the

media had a significantly positive influence on lytic enzyme production in comparison to

the control (NB+chitin). However, soybean meal, urea and NH4Cl were not only unable to

increase the enzyme production but also induce all the three enzymes better than the

control. The results identified CSL and KNO3 as the best nitrogen source (Fig.4.9).

Supplementation of CSL and KNO3 (1.0 g/l) in the media increased chitinase production by

4.87 and 6.09 folds, respectively; glucanase production by 3.51 folds with CSL; cellulase



production by 1.85 and 1.26 folds respectively. Statistical analysis by One-way ANOVA

indicated significant increase in the production of all the three lytic enzymes in the presence

of CSL over control (Table 4.6).

The results obtained in the present study are in agreement with Ray et al. (2007) who

reported that organic nitrogen sources were found to be more suitable for optimizing

cellulase production by Bacillus subtilis and Bacillus circulans than inorganic sources. Urea

was found to be the suitable nitrogen source for chitinase production by Paenibacillus sp.

D1. Gohel et al. (2006) has also reported urea as an important constituent for chitinase

production by Pantoea dispersa. Plackett–Burman studies revealed yeast extract as the most

significant component with a positive effect on chitinase production by Paenibacillus sp.D1.

Earlier report on chitinase production by Paenibacillus sabina strain JD2 showed yeast

extract had negative effect (Patel et al., 2007). It was also reported that yeast extract and

peptone have significant effect on cellulase production (Li et al., 2008).

In Geobacillus sp. it has been reported that optimizing the culture conditions

and addition of yeast extract and ammonium sulfate resulted in two fold increase in

cellulase production (Deepmoni et al., 2011). In this study, both organic and inorganic

nitrogen sources enhanced mycolytic enzyme production by B. subtilis.



Fig. 4.9. Effect of nitrogen sources on mycolytic enzyme production by B.subtilis.

Table 4.6. ANOVA for nitrogen effect on mycolytic enzymes production by B.subtilis


Mean square 8.55 2.38 1.49 0.425 15.96 0.59

F-value 3.59 3.51 27.05

P(5%) 3.11 (S)*

2.65 (S)*

3.11(S)*



4.4.5.3 Surfactants and metal ions

Effect of various metal ions and surfactants tested is summarized in Figure 4.10 and

4.11 respectively. The enzyme production showed varied response to the supplementation of

different metal ions. Addition of CaCl2 and CoCl2 enhanced chitinase production

significantly in comparison to the control as evident from the statistical analysis, by 3.74

and 3.47 folds, respectively. But the same was repressed by MgCl2, CuSO4, HgCl2 and

FeSO4. Glucanase production was positively influenced by CoCl2, FeSO4 and CaCl2 with

6.06, 8.19 and 2.13 fold increase, respectively, and repressed by MgCl2, CuSO4, ZnSo4 and

FeSO4; and cellulase production was increased by addition of FeSO4 (1.66), CuSO4 (1.53)

and MgSO4 (1.25) folds, respectively, and repressed by ZnSO4. Overall, addition of CoCl2

and CaCl2 enhanced the production of all the three mycolytic enzymes by B.subtilis.

Effect of metal ions on chitinase production by bacteria has not been studied in

detail. Chitinase production by Paenibacillus sp. D1 was enhanced by FeCl3 addition (Singh

et al., 2010). This is in contrast with the report by Patel et al. (2007) on Paenibacillus

sabina in which CaCl2 significantly affected chitinase production. Gohel et al. (2006) has

also reported CaCl2 as important media constituent for chitinase production by Pantoea

dispersa. Ghanem (1992) found that addition of FeCl3.6H2O highly induced chitinase

production by Bacillus amyloliqefaciens. Felix and Marco (2007) have reported that only

FeCl3 had a significant impact on cellulase production but all other metal ions did not

impact the production.



Fig. 4.10: Effect of metal ions on mycolytic enzyme production by B.subtilis.

Table 4.7. ANOVA for metal ions effect on mycolytic enzymes production by B.subtilis


Mean square 5.63 0.43 6.71 0.11 6.05 0.35

F-value 13.08 61 17.29

P(5%) 2.65(S) 5.99(S) 2.51(S) *(S)- Significant



Fig. 4.11: Effect of surfactants on mycolytic enzyme production by B.subtilis.

Table 4.8. ANOVA for surfactants effect on mycolytic enzymes production by

B.subtilis


Mean square 9.16 0.985 9.48 0.3 15.88 0.609

F-value

9.29 31.6 26.07

P(5%) 3.48(S)*

3.48(S)*

3.48(S)*

*(S)- Significant



Detergents Triton X100, CTAB, Tween 20 and Tween 80 had positive effect on

chitinase production, while SDS had an inhibitory effect, TritonX100, Tween 20 and Tween

80 positively influenced glucanase production while CTAB and SDS had slight inhibitory

effect. Cellulase production was positively influenced only by Triton X100 addition while

all other detergents negatively affected the production. Triton X100 was identified as the

most significant surfactant supplement with an increase in chitinase production by 5.49

folds; glucanase by 7.39 folds and cellulase by 1.91 folds, respectively.

Effect of surfactants on chitinase production has not been much studied. Vaidya et

al. (2001) had reported positive effect of non ionic detergents on chitinase production by

Alcaligenes xylosoxydans. Triton X 100 was identified as best surfactant supplement for

chitinase production by Aeromonas sp. (Al-Ahmadi et al., 2008). Surfactants are known to

alter the porosity of cell membrane resulting in leakage of enzyme into the external medium.

Addition of surfactant in the medium can, therefore, improve the enzyme production. Felix

and Marco (2007) have reported that the detergent SDS and the reducing agent b-

mercaptoethanol did not drastically affect the production of cellulase by T.harzianum.

4.4.5.4 pH

The influence of pH and temperature on lytic enzyme production was studied by

setting the pH range between 3-9 and incubating at two different temperature conditions

and comparing with a control with pH & and temperature 30°C . Maximum production of all

three enzymes by B.subtilis was observed when initial pH of the medium was set at 7.0 (Fig.

4.12). Chitinase production was seen in the pH range from 4-7 and considerably dropped at

alkaline range. Glucanase production was observed in the range pH 4-9 with increase in

production at the alkaline range. Cellulase production was also observed in the pH range of

4-7 with optimum production at pH 7. Statistical analysis of the results by ANOVA



indicated that the P value was significant at P≤0.05 level of significance and pH 7 was

found to have the most positive influence on lytic enzyme production (Table 4.9).

The pH of the culture medium is playing important role in chitinase production.

Majority of the bacteria reported to produce maximum level of chitinase at neutral or

slightly acidic pH and whereas fungi mostly secret it in acidic conditions (Ulhoa and

Peberdy, 1991; Kovacs et al., 2004; Zhang et al., 2004; Sharaf, 2005). In contrast, B.

laterosporus MML2270 produced highest chitinase at pH 8.0 and interestingly it failed to

produce chitinase at pH 4.0. Similar optimum pH of 8.0 for chitinase production was

reported in B. pabuli K1 (Frandberg and Schnurer, 1994). In contrast to our observation,

Song et al. (1985) reported optimal cellulase production at pH 9 using Clostridium

acetobutylicum. Cellulase production has also been reported at acidic (Hagerdal, 1979) and

neutral pH (Spreinat et al., 1990). Souichiro et al. (2004) reported optimum initial pH for

growth and cellulose degradation of C. straminisolvens sp. at pH 7.5.



Fig. 4.12: Effect of medium pH on mycolytic enzyme production by B.subtilis.

Table 4.9. ANOVA for pH effect on mycolytic enzymes production by B.subtilis.


Mean square 11.10 0.293 6.66 0.211 18.89 25.13

F-value

37.88 31.56 0.75

P(5%) 3.48(S)*

3.48(S)*

3.48(S)*

*(S)- Significant



4.4.5.5 Effect of incubation temperature on mycolytic enzyme production

The production of mycolytic enzymes was evaluated at 30°C and 37

°C. The

production of all the three enzymes was detected at both the temperatures (30 and 37°C),

however, at 30°C the production was significantly more. At 37

°C all the three enzymes

possessed approximately similar activity. B. laterosporus produced high chitinase activity at

35oC, in which good bacterial growth has also been recorded. Paenibacillus sp. D1 isolate

exhibited chitinase production over a wide temperature (25 - 45°C) and pH (6 - 9) range

(Singh, 2010). Kavi Karunya et al. (2011) observed chitinase production in the temperature

ranging from 25oC to 40

oC. However, maximum chitinase activity was reported at 35

oC.



Fig. 4.13. Effect of incubation temperature on mycolytic enzyme production by B.

subtilis.

Table 4.10. ANOVA for temperature effect on mycolytic enzymes production by

B.subtilis.


Mean square 10.67 0.93 1.19 0.07 73.43 0.65

F-value

11.51 17 112.96

P(5%) 7.71(S)*

7.71(S)*

7.71(S)*

*(S)- Significant



Fig. 4.14: Comparison of mycolytic enzyme production by B.subtilis in optimised and

unoptimised media



ANOVA (One-way) was performed for each of the media components affecting the

production of the three mycolytic enzymes by B.subtilis. p-value was found to be significant

for carbon, nitrogen, metal ions, surfactants, pH and temperature (P≤0.05) indicating

significant influence of these varying factors on enzyme production.

Optimized media containing (g/L): CMC, 10; Corn Steep Liquor, 1; Beef extract, 3;

NaCl, 5; CaCl2(50mM), Triton X 100(5mM), pH, 7, showed a significant increase in the

activities of all the three enzymes; chitinase (2 folds); β-1,3-glucanase (1.5 folds) and

cellulase (2 folds) respectively (Fig. 4.14).

4.5 Mechanism of antagonism

Many mechanisms operate in the biocontrol of fungal phytopathogens such as

mycoparasitism, antibiosis and predation and so on. However, to characterize the

antagonistic mechanism by the biocontrol agent, a mutant with loss/ enhancement of

antagonistic phenotype can be developed. In an attempt to assess the role of mycolytic

enzymes of B. subtilis as the molecular basis of antagonism, chemical and physical

mutagenesis was employed to get the desired mutants.

4.5.1 EMS mutagenesis

EMS mutagenesis yielded 60 isolated mutants on NA plates, of which 3 (M3, M4,

and M24) mutants showed loss of antagonism against C. gloeosporioides and 6 mutants

(M8, M21, M22, M57, M58, and M59) exhibited increased antagonism against C.

gloeosporioides. The remaining mutants did not show any difference in their antagonistic

property (Plate 4.5). These mutants were studied for their mycolytic enzyme activities under

shake flask conditions. Mutants M4 and M8 showed a complete loss of chitinase and β-1,3-

glucanase activity; mutants M21 and M22 showed a complete loss of chitinase activity;

mutants M3, M24, M58, and M59 showed decrease in chitinase activity; mutant M24



showed a complete loss of β-1,3-glucanase activity. All the mutants except M21showed

varied levels of increase in cellulase levels. These mutants were further checked for their

antifungal activity using C. gloeosporioides mycelia and it was found that M8, M22, M57,

M58, and M59 showed increased hydrolytic activity with concomitant increases in one or

more mycolytic enzyme levels as compared to wild type (Fig. 4.15). Of all these mutants,

M57 showed 36-fold and 4.71-fold increases in β-1, 3-glucanase and cellulase activities,

respectively, with a concomitant 1.95-fold increase in hydrolytic activity, followed by M59

with 5.68-fold and 1.57-fold increases in β-1,3-glucanase and cellulose activities,

respectively, with a 2.23-fold increase in hydrolytic activity as compared to the wild type B.

subtilis strain. The mutant M24 exhibited a complete loss of β-1, 3-glucanase and decrease

in chitinase with concomitant decreases in levels of hydrolytic activity with C.

gloeosporioides mycelia as compared to the wild type strain (Fig. 4.15). All these

observations clearly indicated the mycolytic enzyme mediated antagonism of this strain.

ANOVA has been performed for hydrolytic enzyme production by the wild type and

mutants of B. subtilis. P-value was found to be very low at both P = 0.05 and P = 0.01,

which indicated that there are significant differences in mycolytic enzyme production

between the strains (Table 4.11).

In a similar study, Kandasamy and Saleem (2002) showed the role of the Bacillus

strain BC121 in suppressing the fungal growth in vitro when studied in comparison with a

mutant of that strain that lacks both antagonistic activity and chitinolytic activity. Lorito et

al. (1993) reported chitinolytic enzymes contributing to the ability of Trichoderma sp. to act

as biocontrol agents.

Strain improvement can generally be described as the use of any scientific

techniques that allow the isolation of cultures exhibiting a desired phenotype. The

technology has been utilized for more than 50 years in conjugation with modern submerged



culture fermentations (Victor and Graham, 1999). EMS is a well-known mutagenic agent,

whose mode of action is attributed to alkylation at nitrogen position 7 of guanosine of the

DNA molecule, leading to transversion or transition type of mutations (Freese, 1961).

Graeme-Cook et al. (1991) reported that high antibiotic production by two T. harzianum

mutant strains, BC10 and BC63, increased inhibition of hyphal growth of R. solani and P.

ultimum. Bapiraju et al. (2004) reported mutation induced enhanced lipase production from

Rhizopus sp. using UV radiation and NTG. Kadam et al. (2006) successfully employed UV

mutagenesis to improve a strain of Lactobacillus delbruekii for lactic acid production.

Successful use of EMS in induced mutations has been reported for many bacterial strains

(Shantamma et al., 1972; Haq et al., 2009). Increase in chitinolytic enzyme production was

reported in Pseudomonas stutzeri YPL M26 after UV and NTG mutagenesis Mutagenesis

increased enzyme production successfully in Trichoderma (Mandels et al., 1971) and some

other fungi in an industrial process (Mantyla et al., 1998). G. virens mutants have shown

differences in their ability to produce the antibiotic gliovirin (Howell et al., 1983).

Mutagenesis altered the production of antibiotics and mycolytic enzymes in biocontrol

agents in T. viride (Pandey et al., 2000).



Plate 4.6. Dual plate assay showing different antagonistic levels of EMS mutants

against C.gloeosporoides on PDA plates. Plate A & C showing Mutants 21, 22, 23 & 24

and mutants 1, 2, 3 & 4, respectively with varied levels of inhibition; Plate B showing

mutants 57, 58 & 59 with increased inhibition.



Fig. 4.15. Mycolytic activity of Bacillus subtilis Wild type and EMS mutants. Values

are mean ± SE of three replicates.

Table 4.11. ANOVA for hydrolytic enzymes production by wild type and EMS mutant

strains of B.subtilis.

Source of

variation

Degree of

freedom

Sample

square

Mean

Square

F-statistics P-value

Between

Samples

9 291.92 32.43 853.42 2.45(S)*

Within

Samples

20 0.76 0.038



4.5.2 UV mutagenesis

UV mutagenesis yielded 61 mutants on NA plates. Out of 61 putative mutant

colonies tested, 6 showed no antagonistic property against C.gloeosporioides (Plate 4.6).

These mutant isolates also did not grow over the mycelial mat and were named as M17,

M22, M23, M27, M28 and M30.

The induction profile of the Bacillus subtilis was checked with autoclaved

Colletotrichum gleosporiodes mycelium used as the carbon source in the medium. Data

represented in Figure 4.16, showed that the lysis of dead mycelia of C.

gloeosporioides was very efficient by Bacillus subtilis (BC2). Appreciable levels of all the

three enzymes were observed in presence of the autoclaved mycelia – chitinase (2.8 U/mL

by day 1; 2 U/mL by day 2and 1.4U/mL by day 3; ), β-1, 3 Glucanase (2.8 U/mL by day 1;

3.2 U/mL by day 2; 2.7U/mL by day 3 and 1U/mL by day 4) and β-1, 4 Glucanase (6U/mL

by day1; 12U/mL by day 2; 13.21 U/mL by day 3 and 8U/mL by day 4) suggesting the

possible role of these enzymes in mycoparasitism.

Further the mutants were studied for their mycolytic enzyme activities under shake

flask conditions. All the mutants showed significant loss of all three mycolytic enzyme

activities (Fig. 4.16). The mutants also exhibited low levels of hydrolytic activity with

C.gloeosporioides mycelia as compared to the wild type strain indicating clearly the

mycolytic enzyme mediated antagonism of this strain (Fig. 4.17).

Moataza Saad (2006) also reported varied levels and types of mycolytic enzymes by

different Pseudomonas strains with different pathogens like P.capsici and R.solani.

To test the antifungal activity of the Bacillus strain BC2, dual liquid culture method

was employed. The differences in dry weights between the fungal cultures grown with BC2

strain or the mutant strains or the control culture grown without any bacterium were



recorded according to Broekaert et al. (1990). There was almost 100% reduction in dry

weight of the culture grown with BC2 strain when compared to the control. There was very

little reduction in dry weight of the culture when grown with the mutant strains. This clearly

shows that the reduction in dry weight of the fungus when grown with the BC2 strain is due

to the antifungal activity of this bacterium. The mutant strains which had lost the antifungal

activity could not reduce the dry weight of the fungus.

The ANOVA (Analysis of Variance) was performed for all three mycolytic enzymes

and hydrolytic activity by the Wild type and mutants of B.subtilis. p-value was found to be

very low at both p<0.05 and p<0.01, which indicated that there is significant difference in

mycolytic enzyme and hydrolytic activity between the strains and the wild type (Table

4.12).

In a similar study Kandasamy and Saleem (2002) showed the role of the Bacillus

strain BC121 in suppressing the fungal growth in vitro when studied in comparison with a

mutant of that strain, which lacks both antagonistic activity and chitinolytic activity.

Another study by Balasubramanian et al. (2010) reported that on testing the biocontrol

efficacy of the mutants and wild strain against phytopathogens such as Fusarium

oxysporum, Bipolaris oryzae, Rhizoctonia solani and Alternaria sp. by dual culture assay on

PDA medium the UV H11 mutant and adapted mutant showed increased biocontrol activity

when compared to wild strain. Balasubramanian et al. (2010) further reported that the

antagonism of these two mutants with F. oxysporum, R. solani, B. oryzae and Alternaria

sp. were varied and could be related with lytic enzyme production with fast growing ability.

However, Lorito et al. (1993) reported chitinolytic enzymes contributing to the ability of

Trichoderma sp to act as biocontrol agents. Graeme-cook et al. (1991) reported that high

antibiotic production by two T. harzianum mutant strains, BC10 and BC63, increased

inhibition of hyphal growth of R. solani and P. ultimum, while Papavizas et al. (1982) have



shown UV-induced benomyl resistant mutant to suppress the saprophytic activity of

R. solani more effectively than the wild strain (Papavizas et al., 1982). Dunne et al. (1997)

employed similar techniques involving Tn5 insertion mutants and subsequent

complementation to demonstrate that biocontrol of Pythium ultimum in the rhizosphere of

sugar beet by Stenotrophomonas maltophila was due to the production of extracellular

protease. Similarly, Preecha et al. (2010) used UV mutagenesis to suggest that the ability of

B. amyloliquefaciens KPS46 to reduce bacterial pustule severity on soybeans is associated

with the production of a lipopeptide surfactin encoded by srfAA.



Plate 4.7. Bacillus strain mutants M17, M22, M23, M27, M28 and M30 not showing

inhibition to Colletotrichum gloeosporioides on PDA.

Fig. 4.16. Mycolytic activity of Bacillus subtilis Wild type BC2 and UV mutants. Values

are mean ± SE of three replicates.



Fig. 4.17: Comparison of hydrolytic activities of the UV mutants and the wild strain

BC2.

Table 4.12 ANOVA for mycolytic enzyme production by wild type and UV mutants of

B.subtilis

Chitinase β 1,3glucanase β 1,4

Glucanase

Hydrolytic Assay

Mean square 2.796

0.175

2.344

0.044

8.999

0.204

6.352

0.864

F-value 15.929 52.752 44.017 7.348

P (5%) 2.9 2.37 2.45 3.97



4.5.3 Sensitivity of the culture supernatant of B. subtilis to proteolytic enzymes, TCA

and heat treatment

After evaluating the mechanism of action through mutagenesis, to further establish

the nature of antibiosis, the sensitivity of the WT crude culture filtrate of B.subtilis BC2 was

tested with TCA, heat and proteolytic enzymes (Table 4.13). The properties of the treated

filtrate were evaluated in terms of lytic enzyme activity and antagonistic ability against the

pathogen. The treatment of the culture filtrate with TCA resulted in a total loss of residual

activity of the lytic enzymes. The treated filtrate also demonstrated a loss of antifungal

activity against the pathogen. The culture filtrate when subjected to heat treatment, showed

a gradual reduction in lytic enzyme activity with a concomitant reduction in antagonism.

Trypsin treatment of the culture filtrate also resulted in a similar loss of residual lytic

activity and antifungal activity. All these results indicated loss of antifungal activity upon

heat, TCA and Trysin treatment, hence establishing the proteinaceous nature of the

antifungal compound and majorly a lytic enzyme mediated antibiosis. The study was in

accordance with the work by Tang et al. (2012) proving the protein mediated antagonism of

B. licheniformis BS-3. In contrast to the present study, Tendulkar et al. (2007) reported that

the antifungal activity of Bacillus licheniformis BC98, on phytopathogen Magnaporthe

grisea was highly stable at extreme pH and temperatures, and also after treatment with

pepsin, trypsin and different detergents thereby indicating the lipopeptide nature of the

antagonistic compound.



Table 4.13. Sensitivity of culture supernatant of Bacillus subtilis to heat, protease and

TCA . Antifungal assay determining the percentage inhibition.

Treatments Percentage inhibition

Heat

CONTROL (30°C)

50° C for 20 min

60° C for 20 min

70° C for 20 min

80° C for 20 min

90° C for 20 min

Autoclaving (121° C for 20 min)

84±6.02

84±6.02

45±6.02

0

0

0

0

Enzyme

Trypsin (1 mg mL-1

)

0

TCA 0



4.6 Assessing antagonistic potential of B.subtilis against selected pathogens in chilli

seeds

4.6.1 In vitro seedling assay

The results of the In vitro seedling assay of Bacillus subtilis treatment of chilli seeds

on seed germination, incidence of pathogen attack in the seedling by C. gloeosporioides

and R. Solani is presented in Figure 4.18. Treatment of the chilli seeds with Bacillus subtilis

culture, showed a germination index 92% similar to the untreated seeds. Treatment of the

seeds with C.gloeosporioides revealed a complete inhibition of germination, but with

R.solani a germination percentage of only 10% was observed. However, co-inoculation of

the seeds with the pathogens and B.subtilis increased the germination percentage by 60%.

Seed treatment with C.gloeosporioides exhibited a disease incidence of 75%. However,

treatment of the seeds with C.gloeosporioides and B.subtilis showed 63% reduction in

disease incidence. Similarly coinoculation of R.solani and B.subtilis exhibited a disease

reduction of 73.3% in comparison to the seed treated with the pathogen alone where the

disease incidence was 83.3%. The germination percentage and reduction in disease

incidence was significantly high in B.subtilis treated seeds in comparison to the untreated

control and pathogen inoculated seeds. This is in agreement with Kamil et al. (2007) who

reported that the seed coat treatment of sunflower seeds with Bacillus licheniformis,

induced high reduction in percentage of infection of R. solani damping off (from 60 % to 25

%) as compared with the pathogen alone. Our observations also comply with these reports.

Sundaramoorthy and Balabhaskar (2012) have reported similar observation with tomato

seeds bacterised with a consortia of B. subtilis and Pseudomonas flourescens.



Fig. 4.18. In vitro chilli seed bacterisation assay to determine germination percentage

and disease incidence in the presence of the respective fungal pathogens- C.

gloeosporioides and R. solani (single and coinoculation with B.subtilis) and B. subtilis

(single and coninocualtion with repective pathogens).



4.6.2 In Vitro Root Colonization

The ability of B.subtilis to colonize chilli roots was evaluated after 15 days of

treatment. The counts reached 2x105cfu/cm of the root and were found to be significantly

higher than the uninoculated control. In the present study, in vitro root colonization

demonstrated that after fifteen days of germination, the bacterial cell counts obtained from

the roots had increased from 102 to 4.5 x 10

6 cfu/cm root for B. subtilis, as compared to

the control where counting was of 104 cfu/cm root length. The initial inoculum level was

100 cfu/ml. Root colonisation is the delivery system of beneficial microbes and their

products. This is similar to the in vitro root colonization study by Jedabi and Awatif (2009)

who demonstrated that after four days of germination, the bacterial cell counts obtained

from the roots had increased by 16.9x105 cfu/cm root for B. subtilis, by 0.4x10

5 cfu/cm root

for B. licheniformis and by 16.1x105 cfu/cm root for B. cereus as compared to the control

where counting was of 100 cfu/cm root length. The effective colonisation of chilli roots by

B. subtilis might have contributed to their capability to inhibit infection of the roots by

R.solani and reduce root rot. Similar reports on ability of heightened root colonisation and

reduction in disease incidence and growth promotion have been reported by several

researchers (Bochow et al., 1995; Marten et al., 2000; Bais et al., 2004; Basha and

Ulaganathan, 2002).

4.6.3 Shelf life stability of the antagonist B.subtilisin Talc/Lignite based formulation:

Shelf life is very important property of any biocontrol agents for long term storage

of formulations. At the time of storage the cfu count was 2.5 to 3 x 108

cfu/g. In order to

determine the shelf life of B.subtilis in talc and lignite formulation, the present study was

conducted for a period 180 days at two temperatures (30±2°C and 4

°C). The results

representing the population statistics (cfu/g) is depicted in Figure 4.19. Both the

formulations were capable of maintaining the viability for six months which is generally



required for any bioinoculant. In the lignite formulation, there was a gradual decline in the

population. However, at 60 and 90 days of storage the fall in its level of cfu. was not

significant but after 120 days of storage the cfu drastically reduced under both storage

conditions. In the talc formulation, though there was a gradual decline, it was not very

significant. Under both the storage temperatures, the viability of the formulation was well

preserved for 180 days. Population level of the antagonist was stable till the 180th

day with

1.6 x 108

and 1.9 x 108 at 30

°C and 4

°C respectively in talc. Population level in lignite was

stable till 150th

day with 1.5 x 108

and 1.3 x 108

respectively. Talc based strain mixtures of

Bacillus sp. have been found to be effective against rice sheath blight and increased plant

yield under field conditions than the application of individual strains (Nandakumar et al.,

2001). Talc and peat based formulations of P. chlororaphis and B. subtilis were prepared

and used for the management of turmeric rhizome rot (Nakkeeran et al., 2004). Salaheddin

et al. (2010) have reported a decline in shelf life of B.subtilis talc formulation after 60 days

of storage. Due to the ease of usage and better shelf life, talc based formulations were used

for further work.



Fig. 4.19 Viability studies of B.subtilis formulation



4.7 Assessing antagonistic potential of B. subtilis against selected pathogens in chilli

seedlings under glass house conditions

4.7.1 Soil analysis

Routine soil testing is done to determine nutritional status of soils and fertilizer

needs. Tests are available to evaluate nearly all elements essential for plant growth. In the

present study, the soil analysis (Table 4.14) indicated that this soil is suitable for chilli

cultivation without further addition of any secondary and micronutrients as all these

nutrients were optimally present . The analysis is in agreement to soil analysis reports

(Nirmal et al., 2003). Addition of N, P and K was essential for good growth of chilli crop.

Table 4.14. Physico chemical Properties of experimental soil.

Soil classification Typic Haplustert

Soil Texture Loamy sand

Bulk density 1.41 g cm3

pH 5.8

E.C 0.27 dS m-1

Org. C 5.24 g kg-1

Mineralizable N 242 kg ha-1

Bray’s P 27.3 kg ha-1

Exchangeable K 149 kg ha-1

Exchangeable Ca 1240 kg ha-1

Exchangeable Mg 278 kg ha-1

Extractable S 11.36 ppm

DTPA Zn 0.620 ppm

DTPA Cu 0.371 ppm

DTPA Mn 4.32 ppm

DTPA Fe 56.4 ppm



4.7.2 Evaluation of the application methods of B.subtilis formulation

The results of soil treatment of B. subtilis on plant growth promotion, enhancement

of yield and suppression of C. gloeosporioides and R. solani is given in Table 4.15. The

growth parameters viz., plant height root length, shoot length, fresh weight, dry weight and

yield (per plant) were found significantly higher in B. subtilis inoculated treatments (T1) in

comparison to the untreated control (T12). Among the treatments, the plant height (70.33

cm), root length (26.0 cm), shoot length (44.33 cm) fresh weight (59.67 g), dry weight (30.5

g) and yield (51.39 g / plant) were superior in B. subtilis treatment (T1) when compared to

all other treatments (Fig. 4.20). The treatment of the plants with either of the pathogens

brought down all the growth parameters by 2 fold with respect to R. solani and 1.5 fold in

the case of C.gloeosporioides (T9 & T7). Among the two pathogens tested, the yield was

found significantly lower in R. solani (25.71g) as compared to C. gloeosporioides (32.05g).

Co-inoculation of either of the pathogens with B. subtilis recorded significantly

higher growth parameters and yield in comparison to pathogen treatment alone. Similarly,

treatment with standard fungicides (Carbendazim or Chlorothalanil) (T8 & T11) for the

respective pathogens C. gloeosporioides or R. solani recorded plant growth parameters

comparable to that of the untreated control (T12). Concerted treatment of the plants with

respective fungicides and B. subtilis also exhibited a significant increase in all the

parameters thereby supporting the compatibility of the BCA with the fungicides (T4 & T5).



SOIL TREATMENT

Table 4.15. Efficacy of B.subtilis at reduction of anthracnose and root rot disease in

chilli caused by C. gloeosporioides and R. solani in pot trials using soil treatment.

Control plant indicates healthy plants not inoculated with pathogen. Each replicate

consisted of a single pot with two plants per pot.

Means followed by the same alphabet(s) in each column are not significantly different based

on Duncan’s multiple range test at p ≤ 0.05.

Treatments Plant

Height

(cm)

Root

length

(cm)

Shoot

length

(cm)

Fresh

weight

(g/Plt)

Dry

weight

(g/Plt)

Yield

(g/Plt)

Disease

incidence

(%)

B.subtilis(T1) 70.33a 26.00

a 44.33

ab 59.67

a 30.50

a 51.39

a -

B. subtilis +

C.gloeosporioides

(T2) 67.67 ab

25.00 ab

42.67abc

59.67a 29.50

a 48.02

abc

0

B.subtilis +

R. solani (T3) 61.33cd

20.67def

39.00c 51.83

bc 21.33

b 42.11

de

0

Carbendazim +

B.subtilis(T4) 68.17 ab

23.00bc

45.17a 52.67

bc 17.67

c 49.37

ab

-

Chlorothalonil +

B.subtilis.(T5) 64.50bc

22.00cde

42.83ab

53.83b 20.00

bc 45.09

bcd

-

Carbendazim +

C.gloeosporioides

(T6) 59.50de

18.83f 40.67

bc 49.83

bc 17.67

c 40.81

de

0

C.gloeosporioides

(T7) 47.33f 15.00

g 32.33

d 30.17

d 13.17

d 32.05

g

50

Carbendazim (T8) 61.00

cd 20.00

ef 41.00

bc 51.17

b 18.50

c 42.98

cde

-

R.solani(T9)

38.33g 13.17

h 25.17

e 29.00

d 11.00

d 25.71

h

66

R.solani+

Chlorothalonil

(T10) 55.75e 22.58

cd 33.17

d 47.00

c 22.00

b 39.23

ef

0

Chlorothalonil

(T11) 56.50e 21.50

cde 35.00

d 52.33

bc 20.33

bc 39.13

ef

-

Control

(untreated)(T12) 61.00cd

16.67g 44.33

ab 49.00

bc 31.00

a 35.40

fg

-

SEM± 0.09

0.08

0.11

0.15 0.18

0.15

F **

** ** ** **

**

CD @ 0.01 0.35

0.32 0.40 0.55 0.52

0.56

CV% 2.98

4.54 4.19 5.16 7.37 5.66



Fig. 4.20. Post harvest evaluation of plant growth parameters of soil treatment



Similarly, seed application results of Bacillus subtilis on plant growth promotion,

enhancement of yield and suppression of pathogens, C. gloeosporioides and R. Solani is

given in Table 4.16 . The plant growth parameters such as plant height, root and shoot

length, fresh and dry weight and yield (per plant) were found significantly higher in B.

subtilis inoculated treatments (T1) when compared to untreated control (T12). Evaluation of

the treatments revealed, plant height ( 75.50 cm), root length (26.17 cm), shoot length (

49.33 cm) fresh weight ( 76.83 g), dry weight (34.17 g) and yield (52.41g / plant)

superiority in B. subtilis treatment (T1) alone as compared to all other treatments (Fig.

4.21). Treatment of plants with any one of the pathogens brought about a considerable

reduction in all the growth parameters inclusive of the yield per plant. Among the two

pathogens tested, the yield reduction was found significantly lower in R. Solani (27.67) (T9)

as compared to C. gloeosporioides (31.97) (T7).

However, co-inoculation of either of the pathogens with the biocontrol agent (T2 &

T3) resulted in an increase in all the parameters in comparison to pathogen treatment alone.

Similarly treatment with carbendazim and chlorothalonil (T8 & T11) for C. gloeosporioides

or R. Solani, respectively, resulted in growth parameters comparable to that of the untreated

control (T12). Results of concerted treatment of the plants with respective fungicide and B.

subtilis, supported the compatibility of the BCA with the fungicides.



Table 4.16.Efficacy of B.subtilis at reduction of anthracnose and root rot disease in

chilli caused by C. gloeosporioides and R. solani in pot trials using seed treatment.

Control plant indicates healthy plants not inoculated with the pathogen. Each replicate

consisted of a single pot with two plants per pot.

Means followed by the same alphabet(s) in each column are not significantly different

based on Duncan’s multiple range test at p ≤ 0.05.

Treatments Plant

Height

(cm)

Root

length

(cm)

Shoot

length

(cm)

Fresh

weight

(g/Plt)

Dry

weight

(g/Plt)

Yield

(g/Plt)

Disease

incidence

(%)

B.subtilis (T1) 75.50

a 26.17

a 49.33

a 76.83

a 34.17

a 52.41

a

-

B. subtilis +

C.gloeosporioides

(T2) 66.67b 22.00b

cd 44.67

b 61.50

b 30.83

a 48.04

ab

20

B.subtilis +

R. solani

(T3) 58.83cd

19.83de

39.00c 52.33

d 18.67

bc 43.52

bc

16

Carbendazim +

B.subtilis

(T4) 70.00b 23.50

b 46.50

b 54.83

cd 22.00

b 49.16

ab

-

Chlorothalonil +

B.subtilis(T5) 66.50b 22.17

bc 44.33

b 53.67

cd 16.50

c 47.78

ab

-

Carbendazim +

C.gloeosporioides

(T6) 57.00d 18.00

ef 39.00

c 51.00

d 17.33

c 40.58

c

0

C.gloeosporioides

(T7) 48.83f 16.50

f 34.17

d 31.67

e 11.50

d 31.97

de

80

Carbendazim

(T8) 61.00c 21.17

bcd 39.83

c 53.00

cd 12.67

d 44.60

bc

R.solani

(T9) 37.33g 13.33

g 24.00

f 31.83

e 10.67

d 27.67

e

100

R.solani+

Chlorothalonil

(T10) 49.83f 18.50

ef 31.33

e 49.67

d 18.17

bc 41.65

c

16

Chlorothalonil

(T11) 53.50e 20.83

cd 32.67

de 57.50

bc 16.67

c 33.79

d

-

Control (untreated)

(T12) 61.00

cd 16.67

g 44.33

ab 49.00

bc 31.00

a 35.40

fg

-

SEM± 0.08 0.09 0.07 0.13 0.15 0.15

F ** ** ** ** ** **

CD @ 0.01 0.29 0.34 0.27 0.48 0.58 0.57

CV% 2.42 4.82 2.82 4.29 8.75 5.66



Fig. 4.21. Evaluation of post harvest biometric parameters of seed treatment



The results of foliar spray application of Bacillus subtilis formulation, on plant

growth promotion, enhancement of yield is given in Table 4.17. The growth parameters

such as plant height, root length, fresh, dry weight and yield were found significantly higher

in B. subtilis (T2) inoculated treatments in comparison to the fungicide treatment (T3 & T4)

and untreated control (T1). However, there was no significant difference in shoot length

between the treatments. Among the treatments, the plant height (70.17 cm), root length

(25.67 cm), fresh weight (48.83 g), dry weight (20.5 g) and yield (47.96g / plant) were

observed to be superior in B. subtilis treatment (T2) as compared to all other treatments (T2,

T3 & T4) (Fig.4.22). In this application method, challenge inoculation with the pathogens

was not attempted but the treatments were evaluated for natural infection incitation. There

was no evidence of natural infection with Colletotrichum or Rhizoctonia. However, leaf

spots were noticed in the control. B.subtilis treated plants exhibited overall better plant

rigour and stability in comparison to the fungicide treated plants or the control.



SPRAYING

Table 4.17. Efficacy of B.subtilis at reduction of anthracnose and root rot disease in

chilli caused by C. gloeosporioides and R. solani in pot trials using spray treatment.

Control plant indicates healthy plants not inoculated with the pathogen.

Each replicate consisted of a single pot with two plants per pot. Means followed by the same

alphabet(s) in each column are not significantly different based on Duncan’s multiple range

test at p ≤ 0.05.

Treatments Plant

Height(cm)

Root

length(cm)

Shoot

length(cm)

Fresh weight

(g/Plt)

Dry weight

(g/Plt)

Yield

(g/Plt)

Control (T1) 61.00

b 16.67

c 44.33 41.33

b 19.33

a 36.85

b

B.subtilis (T2)

70.17a 25.67

a 44.50 48.83

a 20.50

a 47.96

a

Carbendazim

(T3) 64.33b 22.00

b 42.33 43.83

ab 19.00

ab 39.11

b

Chlorothalonil

(T4) 60.33b 18.00

c 42.33 37.78

b 15.67

b 37.89

b

SEM± 4.20 4.20 4.12 4.17 4.08

F ** ** NS ** * *

CD @ 0.01 0.41 0.41 0.68 0.54 0.80

CV% 3.11 5.50 6.27 7.50 7.59



The effect of Root dip application of Bacillus subtilis formulation, on plant growth

promotion, enhancement of yield is given in Table 4.18. The evaluation of plant growth

parameters such as root length, fresh weight and yield were found significantly higher in B.

subtilis inoculated treatments (T2) in comparison to the fungicide treatment (T3 & T4) and

untreated control (T1). There was no significant difference in dry weight between the

treatments. Among the treatments, root length (33.33 cm), fresh weight (62.81 g), dry

weight (39.12 g) and yield (72.03 g/plant) observed to be superior in B. subtilis treatment

(T2) as compared to all other treatments (Fig.4.23). However, significantly higher plant

height (78.17cm) and shoot length (45.33cm) was obtained with carbendazim treatment

alone (T3). In this application method, challenge inoculation with the pathogens was not

attempted but the treatments were evaluated for natural infection incitation. Similar to foliar

spray application, there was no evidence of natural infection with Colletotrichum or

Rhizoctonia, but leaf spots were noticed in the control. B.subtilis treated plants retained

better overall plant rigour and stability in comparison to the fungicide treated plants or the

control.



DIP TREATMENT

Table 4.18. Efficacy of B.subtilis in the reduction of anthracnose and root rot disease

in chilli caused by C. gloeosporioides and R.solani in pot trials using dip treatment.

Control plant indicates healthy plants not inoculated with C. gloeosporioides.

Each replicate consisted of a single pot with two plants per pot. Means followed by the same

alphabet(s) in each column are not significantly different based on Duncan’s multiple range

test at p ≤ 0.05.

Treatments Plant

Height

(cm)

Root

length

(cm)

Shoot

length

(cm)

Fresh

weight

(g/Plt)

Dry

weight

(g/Plt)

Yield

(g/Plt)

Control (T1) 58.17c 15.83

c 42.33

b 43.00

b 24.00 26.05

b

B.subtilis(T2) 77.67a

33.33a 44.33

a 62.81

a 39.12 72.03

a

Carbendazim(T3) 78.17a 32.83

a 45.33

a 58.39

a 17.25 65.99

a

Chlorothalonil(T4) 70.33b 28.50

b 41.83

b 56.44

a 22.74 63.86

a

SEM± 4.24 4.23 4.23 3.95 3.21

F ** ** ** ** NS **

CD @ 0.01 0.16 0.22 0.19 1.05 1.99

CV% 1.14 2.54 1.74 8.63 16.38



Fig. 4.22: Evaluation of plant growth parameters of foliar spray treatment

Fig. 4.23: Evaluation of plant growth parameters of root dip treatment



In the present study, Post harvest biometric observations of the application methods

revealed that treatment of the chilli plants with B.subtilis improved the overall growth and

yield as compared to untreated control and the standard fungicides carbendazim and

chlorothalonil, in all the four application methods (Table 4.15- 4.18). Of the four application

methods, dip treatment with Bacillus subtilis formulation, gave significantly higher plant

length (77 cm, 1.26 fold increase); root length (33 cm, 1.86 fold); dry weight (33g, 1.57

fold) and chilli yield (72g, 2 fold) as compared to untreated control (Fig. 4.24). Evaluation

of plant growth parameters for B.subtilis treatment upon challenge inoculation with the

pathogen C. gloeosporioides exhibited plant length (67.7cm, 1.4 fold); root length (25cm,

1.67 fold); dry weight (29g, 2.6 fold) and chilli fruit yield (48g, 1.5 fold) increase when

compared to plants inoculated with C.gloeosporioides alone (T7). These results were similar

in both soil and seed application method (Fig. 4.20 & 4.21). The results also suggested that

B.subtilis treatment was better than the standard fungicide, carbendazim with regard to

promotion of growth and yield (Fig. 4.20 & 4.21). Similarly evaluation of biometric

parameters of B.subtilis treatment on challenge inoculation with R.solani showed plant

height of 61.33 cm (1.6 fold); root length of 20.67cm(1.6 fold); dry weight of 39g(1.6 fold)

and chilli fruit yield of 42g(1.7 fold) increase in comparison to plants inoculated with

R.solani alone (T9). Statistical analysis by ANOVA(One-way) indicated that treatment of

chilli plants with B.subtilis alone or upon co-inoculation with the pathogens resulted in a

significant (P≤ 0.05) increase in plant growth parameters in comparison to the control.

B.subtilis treatment also fared significantly better than the standard fungicide chlorothalonil

and carbendazim in soil, seed and foliar spray application methods. However, in dip

treatment method it was comparable to the carbendazim treatment.

All the four application methods were effective in reducing disease incidence and

maintaining plant rigour. The study showed that B.subtilis treatment proved to be effective



in controlling the disease in both methods of application. A comparison of the data in Table

4.15 & 4.16 (Fig. 4.20 & 4.21) revealed that both seed and soil application methods of

B.subtilis talc formulation tested in the pot experiment were effective in reducing

anthracnose incidence in chilli compared to the control infected with C. gloeosporioides

alone. However, disease incidence was reduced by 50% for soil application and 60% for

seed application. Reduction in disease incidence brought about by B.subtilis treatment was

significant and was comparable to that of carbendazim.

Similarly both the seed and soil application methods were effective in reducing the

Rhizoctonia root rot incidence in chilli in comparison to the challenged control. A 66%

reduction in disease incidence was noticed with soil application but a reduction of 84% was

noticed with the seed application method. Significant reduction in disease incidence was

achieved by B.subtilis treatment and the results were comparable to the standard fungicide

chlorothalonil.

The spray and dip treatment (Table 4.17 & 4.18) with B.subtilis did not witness any

natural infection by C. gloeosporioides or R. solani, though the untreated controls were

infected with leaf spots and showed less rigour in comparison with the plants treated with

the talc formulation (Fig. 4.22 & 4.23).

Salaheddin et al. (2010) have reported a similar reduction in disease incidence of

bacterial blight of cotton using a foliar spray of B.subtilis and Pseudomonas fluorescens

consortia. Similarly Lamsal et al. (2012) have reported 40% reduction in incidence of

anthracnose using different species of Bacillus. Saman Abeysinghe (2007) has reported a

50% reduction in disease incidence of fusarium wilt using B.subtilis CA32. Similarly,

dipping of Phyllanthus amarus seedlings in talc based formulation of B. subtilis (BSCBE4)

or P. chlororaphis (PA23) for 30 minutes prior to transplanting reduced stem blight of P.

amarus (Mathiyazhagan et al., 2004).



The present study demonstrated that the selected bacterial isolates enhance shoot and

root length, fresh biomass and total dry matter in inoculated plants. The chilli plants were

treated with the biofungicides and exhibited disease control due to reduction of disease

incidence. Bacillus species isolated from rhizosphere soil have been reported to be effective

at controlling a variety of soil-borne plant pathogens (Williams et al., 1996). Choudhary and

Johri (2009) elucidated the mechanisms and role of Bacillus species as inducers of systemic

resistance in relation to plant-microbe interactions and identified the pathways involved in

their regulation. Moreover, available reports suggest that specific strains of the species

B.amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B.pumilus, B. mycoides, and B.

sphaericus elicit significant reductions in the incidence or severity of various diseases on a

variety of hosts including greenhouse studies or field trials on tomato, bell pepper,

muskmelon, watermelon, sugar beet, tobacco, cucumber, lobloby pine, and tropical crops

(Kloepper et al., 2004). Ryu et al. (2003) demonstrated the involvement of the production of

volatile compounds 2, 3-butanediol and acetoin in plant growth promotion in Arabidopsis

thaliana by B. subtilis strain GB03 and B. amyloliquefaciens strain IN937a.

Sundaramoorthy and Balabaskar (2012) have shown increased root and shoot length due to

combined application of Pseudomonas sp. and B.subtilis in tomato (Latha et al., 2009) and

chilli (Sundaramoorthy et al., 2012) were also reported.



Fig. 4.24: Evaluation of plant growth parameters in the best application method



4.8 Post harvest studies for anthracnose

Anthracnose caused by Colletotrichum sp. is a major disease of tropical vegetables

such as chilli (Capsicum annuum L. var. acuminatum Fingerh.) (Hadden and Black, 1989;

Pakdeevaraporn et al., 2005). This disease appears as ripe fruit-rot and die-back (Mehrotra

and Aggarwal, 2003). Ripe fruit-rot is more conspicuous as it causes severe damage to

mature fruits in the field as well as during transit and storage. Hence, in the present study a

preliminary post harvest disease control has been attempted.

The chilli fruits were sprayed with the talc formulation of B.subtilis. The isolate

could control anthracnose in chilli fruits inoculated with the agent alone and challenge

inoculated with the pathogen, when compared to the untreated control. After 10 days of

treatment, the highest percentage of survival of fruit from anthracnose was observed with

the biocontrol agent treated chilli. In the present study, the effect of post harvest inoculation

of B. subtilis (JN032305) formulationon chilli fruits was evaluated. The fruits were

evaluated for increased shelf life and disease incidence. The formulation was significantly

effective in reducing the incidence of anthracnose in chilli fruits caused by C.

gloeosporioides by 65.3% in comparison to the untreated control (55%) and challenge

inoculated control (80%) (Fig. 4.25). Currently, biological control is considered a very

promising alternative to synthetic fungicide in the control of post harvest decay of fruits and

vegetables (Wisniewski and Wilson, 1992). Post harvest chilli fruits are usually preserved

by washing or spraying with chlorinated water at 75–400 ppm chlorine and stored at low

temperature (7–10°C) before shipment (Smith et al., 1998; Suslow, 1997). B. subtilis is an

antagonist against the major post harvest pathogens of stone fruits (Pusey et al., 1988),

pome fruits (Wilson et al., 1993) and citrus fruits (Smilanick and Denis-Arrue, 1992).

Similarly, post harvest application of B.subtilis also controlled post harvest avocado

diseases (Korsten et al., 1995)



Figure 4.25: Post harvest disease management of anthracnose using B.subtilis talc

formulation



SUMMARY AND CONCLUSION

SUMMARY

Agriculture is an important sector of Indian economy as it contributes about 17 % to

the total GDP and provides employment to over 60 % of the population. Indian agriculture

has registered impressive growth over last few decades. Chilli, Capsicum annum L.is an

annual herbaceous vegetable and spice grown in both tropical and sub-tropical regions.

India accounts for 25 % of the world’s total production of chilli. The sustainability of chilli-

based agriculture is threatened by a number of biotic factors particularly wilt diseases,

anthracnose and root rot caused by several pathogens both under pre and post harvest

conditions. The present investigation was directed towards developing Bacillus sp. as

effective biocontrol agents against anthracnose and root rot of chilli and also assessing the

mechanism of antagonism. The study was carried out at the Department of Microbiology,

CPGS, Jain University, Bangalore.

Nine chitinolytic Bacillus sp. isolates were obtained from chilli rhizosphere soil from in

and around Bangalore. One isolate exhibiting broad spectrum of antagonism was selected

and evaluated for direct and indirect growth promotion abilities. Induction and optimisation

studies for the culture conditions were conducted. The mechanism of antagonism of the

isolate was also assessed. The biocontrol potential of the isolate against the fungal

pathogens was assessed under in vitro and pot culture conditions. Reduction in disease

incidence under post harvest conditions also was evaluated. The salient features of the

findings are outlined below.



Out of 9 chitinolytic isolates obtained from chilli rhizosphere, one isolate

numbered 2 exhibited broad spectrum inhibition against nine potent chilli

pathogens and was detected positive for chitinase, glucanases, protease and

lipase.

The isolate was morphologically, biochemically and phenotypically

characterized and identified as Bacillus subtilis (JN032305).

Media optimization identified Nutrient agar as the most suitable basal media

for the growth of Bacillus subtilis. Evaluation of Induction pattern of lytic

enzymes with pure substrates demonstrated the inducible nature of the

enzymes and CMC (1%) as the substrate capable of concerted induction of all

the three enzymes.

Extracellular lytic enzymes was induced significantly when Bacillus subtilis was

grown in media supplemented with autoclaved mycelia of the fungal pathogens

Provision of fungal mycelium as substrate for assay of hydrolytic activity also

revealed the ability of the lytic enzymes to utilize the cell wall of the pathogens

for growth and activity

Optimization studies using one factor approach identified the following medium

components to concomitantly increase all three mycolytic enzyme production

by B.subtilis - CMC (1%, w/v); CSL (0.5%, v/v); CaCl2 (5mM); TritonX 100

(0.1%v/v), pH 7.0, 30°C.

Comparison of process parameters showed that using one factor approach all

three enzyme activities increased- chitinase (2 folds); β-1,3-glucanase (1.5 folds)

and cellulase (2 folds) respectively.



Mutants obtained as a result of UV mutagenesis lacked antifungal activity and

were also found to exhibit significantly decreased or no mycolytic enzyme

activity in comparison to the wild strain, when tested against the fungal

pathogen.

EMS mutagenesis identified hyper producing (M59 mutant with 2.23 folds) and

low enzyme producing mutants with concomitant increased or decreased

antagonism, respectively

Mycolytic enzymes mediated mechanism as one of the major mechanisms of

antagonism of B.subtilis was further demonstrated by heat inactivation,

trypsin and TCA treatment of the crude enzyme extract with loss of antifungal

property

In vitro seedling assay demonstrated reduction of Disease Incidence with both

C. gloeosporioides (65%), R. solani (73%) when co-inoculated with B.subtilis

(JN032305).

B.subtilis exhibited heightened root colonisation of chilli plant, establishing

ecological competence, compatibility with the host plant and the environmental

growth conditions.

Viability studies revealed talc formulation (6 months) to be better than lignite

(5 months) in terms of shelf life and ease of usage.

Pot culture studies evaluated four different application methods with different

treatments for disease control and plant growth parameters.

Co-inoculation with R. solani and B.subtilis reduced the disease incidence by

84 % (seed) and 66% (soil) in comparison to R.solani alone. Similarly, co-

inoculation with C. gloeosporioides and B.subtilis reduced the disease incidence

by 60 % (seed) and 50% (soil) in comparison to C. gloeosporioides alone.



Seed application method was more effective than soil application in reducing

the disease incidence

Evaluation of biometric parameters of plant growth demonstrated B.subtilis

treatment to be the best in comparison to the standard fungicides and untreated

control. Root dip application method was most effective in promoting growth in

terms of increase in plant height, root length and yield in comparison to the

other application methods.

The efficacy of B.subtilis in controlling the anthracnose disease incidence under

post harvest conditions was evaluated and the results revealed 63% reduction

disease incidence (75% to 12%) in comparison to the control inoculated with C.

gloeosporioides alone which showed a disease incidence of 75%.

CONCLUSION

Our investigations have been successful in isolating Bacillus subtilis (JN032305.1) capable

of broad spectrum of antagonism against potent chilli pathogens and ability to produce

mycolytic enzymes inducible with pure substrates as well as fungal mycelium, paving way

for a novel biocontrol agent which would be effective in controlling chilli fungal pathogens

at large and anthracnose caused by Colletotrichum gloeosporioides and R.solani root rot in

particular. The study identified the optimum cultivation conditions for increased and

concerted production of all the three mycolytic enzymes- chitinase, β-1,3- glucanase and β-

1,4- glucanase by, a potential biocontrol agent. Attempt to assess the mechanism of

antagonism using mutagenesis approach resulted in mutants with increased or lowered

antagonism with a concomitant alteration in lytic enzyme activity thereby stressing on

mycolytic enzyme mediated antifungal activity. Although naturally occurring organisms



provide a major source of mycolytic enzymes, genetic improvement plays an important role

in their biotechnological applications. Strain improvement by EMS mutagenesis yielded

mutants with increased mycolytic enzymes activity and higher hydrolytic activity also,

when tested against C.gloeosporoides mycelia, the causative agent of anthracnose

disease.Evaluation of the talc formulation of the biocontrol agent, revealed a shelf life of

over six months. Assessment of the biocontrol potential both at In vitro and pot culture

studies revealed the ability of the BCA to significantly reduce disease incidence of both

anthracnose and root rot and also promote overall plant growth thereby stressing on its dual

role.

4.1 isolation and screening of bacillus sp -...

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