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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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 93
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 94
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 95
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 96
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 97
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 98
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 +
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 99
Fig. 4.1. Phylogenetic tree of the Bacillus subtilis based on the 16S rDNA sequences.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 100
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).
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 101
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).
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 102
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 103
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 104
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
-
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 105
Plate 4.4. Mycolytic enzyme profile of the isolate.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 106
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 107
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 108
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 109
Fig. 4.2: Medium optimization for the production of Chitinase (a), β 1,3 Glucanase (b)
and β 1,4 Glucanase (c).
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 110
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 111
Fig. 4.3: Induction of mycolytic enzymes with pure substrates: Chitinase (a), β 1,3
Glucanase (b) and β 1,4 Glucanase (c)
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 112
Fig. 4.4. Optimization of CMC concentration (%, w/v) for maximum mycolytic
enzyme activity.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 113
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 114
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 115
Fig. 4.5: Induction of hydrolytic enzymes with autoclaved mycelium of
C.gloeosporioides (a) and R.solani (b)
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 116
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).
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 117
Fig. 4.6: Hydrolytic activity of B.subtilis on autoclaved fungal mycelium.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 118
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 119
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)
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 120
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.
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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)*
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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
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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.
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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
Chitinase Glucanase Cellulase
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)*
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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.
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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
Chitinase Glucanase Cellulase
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
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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
Chitinase Glucanase Cellulase
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
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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
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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.
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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.
Chitinase Glucanase Cellulase
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
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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.
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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.
Chitinase Glucanase Cellulase
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
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Fig. 4.14: Comparison of mycolytic enzyme production by B.subtilis in optimised and
unoptimised media
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Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 134
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
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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
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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).
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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.
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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
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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
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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
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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.
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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.
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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
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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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 145
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 146
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 147
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).
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 148
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 149
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 150
Fig. 4.19 Viability studies of B.subtilis formulation
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 151
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 152
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).
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 153
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 154
Fig. 4.20. Post harvest evaluation of plant growth parameters of soil treatment
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 155
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 156
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 157
Fig. 4.21. Evaluation of post harvest biometric parameters of seed treatment
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 158
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 159
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 160
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.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 161
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 162
Fig. 4.22: Evaluation of plant growth parameters of foliar spray treatment
Fig. 4.23: Evaluation of plant growth parameters of root dip treatment
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 163
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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 164
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).
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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.
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Fig. 4.24: Evaluation of plant growth parameters in the best application method
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Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 167
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)
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Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 168
Figure 4.25: Post harvest disease management of anthracnose using B.subtilis talc
formulation
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Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 169
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.
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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.
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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.
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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
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 173
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.