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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 28
REVIEW OF LITERATURE
Plant diseases need to be controlled to maintain the quality and abundance of food,
feed and fibre produced by growers around the world. Different approaches may be used to
prevent, mitigate or control plant diseases. Beyond good agronomic and horticultural
practices, growers often rely heavily on chemical fertilizers and pesticides. Such inputs to
agriculture have contributed significantly to the spectacular improvements in crop
productivity and quality over the past 100 years. However, the environmental pollution
caused by excessive use and misuse of agrochemicals, as well as fear-mongering by some
opponents of pesticides, has led to considerable changes in people’s attitudes towards the
use of pesticides in agriculture. Today, there are strict regulations on chemical pesticide use,
and there is political pressure to remove the most hazardous chemicals from the market.
Additionally, the spread of plant diseases in natural ecosystems may preclude successful
application of chemicals, because of the scale to which such applications might have to be
applied. Consequently, some pest management researchers have focused their efforts on
developing alternative inputs to synthetic chemicals for controlling pests and diseases.
Among these alternatives are those referred to as biological controls. A variety of biological
controls are available for use, but further development and effective adoption will require a
greater understanding of the complex interactions among plants, people, and the
environment.
2.1 The Host Chilli
Chilli (Capsicum annum L.), most widely used and universal spice of India, belongs
to the "Solanaceae" family. The nutritive value of chilli is excellent; chillies are rich in
vitamins, especially in vitamin A and C (Ordentlich et al., 1988). Every 100 g of dried pods
yield about 160 calories of energy through 36 g carboydrates, 18 g proteins, 16 and g fat,
480 mg calcium, 3.1 mg. phosphorous, 31 mg iron, 2.5mg niacin, 640 I.U. vitamin 'A' and
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 29
40 mg vitamin 'C' (Palumbo et al., 2005). India has immense potential to grow and export
different types of chillies required to various markets around world. India has produced
around 1014.60 million tonnes of chilli with area of 654 million ha. and productivity 1551
kg/ha during 2005-06 (Source: Directorate of Arecanut and Spices Development; Jagtap et
al., 2012). For the last two decades, many research results have provided convincing
evidence that root health and vigor are directly related to plant productivity. As a
consequence, root disease control has become one of the most challenging research areas in
the context of plant productivity improvement (Benhamou et al., 1990).
2.1.1 Pathogens of chilli
Over 10,000 species of fungi can cause disease in plants. Classes of fungi that
commonly cause diseases in agricultural crops are Plasmodiophoromycetes (cause clubroot
of crucifers, root disease of cereals, and powdery scab of potato), Oomycetes (cause
seedling damping-off, late blight, downy mildews, and white rust disease), Zygomycetes
(cause soft rot of fruit), Ascomycetes and Deuteromycetes (cause leaf spots, blights,
cankers, fruit spots, fruit rots, anthracnose, stem rots, root rots, vascular wilts, soft rot), and
Basidomycetes (cause rust and smut diseases) (Agrios, 2005).
Fungi are ubiquitous; some having beneficial effects on plants, while many others
may be detrimental (Anderson and Cairney, 2004; Ipsilantis and Sylvia, 2007). A decrease
in crop yield as a result of a plant disease caused by a pathogen is a negative effect. Some
fungi are the main pathogens responsible for plant disease and they may cause high yield
losses (Than et al., 2008a and b).
Some of the important fungal diseases of chilli are: Fusarium wilt- of tomato, potato,
eggplant, and chilli, most commonly caused by the soil borne pathogen, Fusarium
oxysporum (Miller et al., 1986); Alternaria fruit rot- causing seed, seedling, leaf and fruit
diseases and post harvest decay of fruits and seeds (Spalding and King, 1981; Singh, 2003);
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 30
Phytophthora Root Rot caused by the soil-borne fungus, Phytophthora capsici, a serious
disease affecting the plants at any growth stage and causing extensive damage on peppers
worldwide (Akgül and Mirik, 2008); Verticillium Wilt- caused by Verticillium dahliae and
V. albo-atrum, inflicts serious physiological changes such as reduction in photosynthesis,
increased transpiration and respiration (Lazarovits, 2000); Colletotrichum anthracnose
affects several hosts worldwide causing extensive damage, among which it severely causes
yield loss of up to 50% in chilli under pre and post havrvest conditions (Pakdeevaraporn et
al., 2005) and Rhizoctonia root caused by Rhizoctonia solani causes damping-off disease of
seedlings as well as root and stem rot in young transplants of chilli (Rini, 2006). However
anthracnose and root rot incite more extensive plant damage in chilli.
Chilli anthracnose usually develops under high humid conditions when rain occurs
after the fruits have started to ripen with reported losses of up to 84% (Thind and Jhooty,
1985). Than et al., (2008) reviewed the causal agents of chilli anthracnose, the disease
cycle, conventional methods in identification of the pathogen and molecular approaches that
have been used for the identification of Colletotrichum species. Pathogenetic variation and
population structure of the causal agents of chilli anthracnose along with the current
taxonomic status of Colletotrichum species were discussed. The sustainability of chilli-
based agriculture is threatened by a number of factors. Anthracnose disease is a major
problem in India and one of the more significant economic constraints to chilli production
worldwide, especially in tropical and subtropical regions.
Rhizoctonia root rot is caused by Rhizoctonia solani [telemorph: Thanatephorus
cucumeris (Frank) Donk], a fungal pathogen of many plant species (Sherf and MacNab,
1986). It infects mature plants and induces root rot, which leads to wilting and death of
chilli plants. R. solani persists in soils and organic debris. Several chilli accessions
belonging to four Capsicum species were evaluated by Muhyi and Bosland (1992) for
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 31
resistance to R. solani. To date, there are no commercially-acceptable chile cultivars that are
resistant to R. solani. Management of this fungal pathogen relies on using high-quality seed
treated with fungicides such as thiram or captan, and avoiding saturated soil conditions. On
chillies, this soil borne fungus can cause seed decay, pre- and post emergence damping-off,
wirestem, root rot, and necrotic spots on the hypocotyl or tap root (Sherf and MacNab,
1986). Biological control of Rhizoctonia diseases has been demonstrated and represents as
an additional strategy that may provide effective and sustainable management (Brewer and
Larkin, 2005). Although biological control of different soil-borne pathogens on chilli
cultivars have been reported by Mao et al. (1998), Ramamoorthy et al. (2002), Sid Ahamed
et al. (2003) and Nakkeeran et al. (2006), biological control of damping-off caused by R.
solani on S. melongena and Capsicum sp. has not been reported.
Viqar Sultana et al. (2006) worked on the efficacy of Ps.aeruginosa in controlling
root rot of chilli. Application of Pseudomonas aeruginosa, a plant growth promoting
rhizobacterium alone or with crustacean chitin, fungicides (benlate/captan) or Paecilomyces
lilacinus (a biocontrol agent) significantly suppressed Macrophomina phaseolina,
Rhizoctonia solani, Fusarium oxysporum and F. solani attacking roots of chilli.
Virgilio Mojica-Marin et al. (2008) evaluated the control of damping-off and root
and stem rot caused by Rhizoctonia solani by 60 strains of Bacillus thuringiensis. 16 isolates
were effective in controlling the pathogen with an inhibition ranging from 37-66%.
Neha and Dawande, (2010) found that Rhizoctonia solani can be controlled by the
antifungal activity of Trichoderma spp. and P. fluorescens. These two antifungal agents
produced a wide variety of enzymes such as beta 1, 4 glucanase, beta 1,3 glucanase,
chitinases.
Rahman et al. (2012) reported that application of culture filtrates of T. harzianum
IMI-392433 (T8) significantly (p=0.05) suppressed the anthracnose fruit rot of chilli
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 32
percentages (94.97 %) when compared to C. capsici treatment alone and improved both
plant growth and yield.
Chanchaichaovivat (2007) evaluated the efficacy of Streptomyces-biofungicide to
control chilli anthracnose caused by Colletotrichum gloeosporioides in pot experiment. The
biofungicides namely NSP-1, NSP-2, NSP-3, NSP-4, NSP-5 and NSP-6 significantly
reduced disease incidence on chilli fruits.
Kabir Lamslal et al. (2012) conducted in vitro and greenhouse screening of seven
rhizobacterial isolates, AB05, AB10, AB11, AB12, AB14, AB15 and AB17 to investigate
the plant growth promoting activities and inhibition against anthracnose caused by
Colletotrichum acutatum in pepper. According to identification based on 16S rDNA
sequencing, the majority of the isolates were identified as members of Bacillus and a single
isolate as Paenibacillus. The isolates were able to exhibit varying degrees of antagonism
against the pathogen.
Huang et al. (2012) tested the antifungal activity of B. pumilus N43 against R.
solani Q1. In dual culture, the mycelium of R. solani Q1 was inhibited by B. pumilus N43
by the production of an antibiotic. Microscopic observation indicated that B. pumilus SQR-
N43 induced hyphal deformation, enlargement of cytoplasmic vacuoles and cytoplasmic
leakage in R. solani Q1 mycelia.
2.2 Biocontrol
The terms “biological control” and its abbreviated synonym “biocontrol” have been
used in different fields of biology, most notably entomology and plant pathology. In Plant
pathology, the term applies to the use of microbial antagonists to suppress diseases as well
as the use of host specific pathogens to control weed populations. In both fields, the
organism that suppresses the pest or pathogen is referred to as the biological control agent
(BCA). The key to achieving successful, reproducible biological control is the gradual
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 33
appreciation that knowledge of the ecological interactions taking place in soil and root
environments is required to predict the conditions under which biocontrol can be achieved
(Whipps, 1997a) and, indeed may be part of the reason why more biocontrol agents are
reaching the market place (Whipps and Davies, 2000; Whipps and Lumsden, 2001).
Significantly, disease suppression can also be achieved by manipulation of the
physicochemical and microbiological environment through management practices such as
use of soil amendments, crop rotations, use of fumigants or soil solarisation. At present,
greatest interest resides with the development and application of specific biocontrol agents
for the control of diseases on seeds and roots and the interaction of these with pathogens and
hosts. The biochemicals used as a pesticide are environmentally safe, selective, specific in
their action, and easily biodegradable. The cost and time of production of biopesticides is
low as compared to chemical based control measures, and they can be used in combination
with other control measures in integrated pest management programs. They seldom have
any effect on non-target organisms, mammals and plants (Boland and Kuykendall, 1998).
Biological control refers to the purposeful utilization of introduced or resident living
organisms, other than disease resistant host plants, to suppress the activities and populations
of one or more plant pathogens (Pal and Gardener, 2006).
Biological control is the deliberate use of one organism to regulate the population size of
a pest organism. There are three main branches of biocontrol (Hoffmann and Frodsham,
1993):-
Classical biological control is the control of pests introduced from another region
through importing specialized natural enemies of the pest from its native range. The
aim is to establish a sustained population of the natural enemies.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 34
Conservation biological control aims to manipulate the environment to favour
natural enemies of the pest.
Augmentation biological control occurs when the number of biological control
agents is supplemented. Inoculation is the introduction of a small number of
individuals of the biological control agent, while inundation is the introduction of
vast numbers of individuals. This over all approach is common when the biological
control agent cannot survive the entire year, or cannot achieve densities high enough
to regulate the pest population. The benefits of biological control are that it can
provide fairly permanent regulation of devastating agricultural and environmental
pests that may be difficult or impossible to manage with more traditional chemical
means.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 35
2.2.1 Mechanism of action
Modes of action include, inhibition of the pathogen by antimicrobial compounds
(antibiosis); competition for iron through production of siderophores; competition for
colonization sites and nutrients supplied by seeds and roots; induction of plant resistance
mechanisms; inactivation of pathogen germination factors present in seed or root exudates;
degradation of pathogenicity factors of the pathogen such as toxins; parasitism that may
involve production of extracellular cell wall degrading enzymes (Table 2.1) (Whipps,
1997a). None of the mechanisms are necessarily mutually exclusive by a single biocontrol
agent. Indeed, for some biological control agents, different mechanisms or combinations of
mechanisms may be involved in the suppression of different plant diseases.Mechanisms of
biocontrol of root and soil borne pathogens are as a result of the direct action of antagonists
on plant pathogens, through antibiosis, predation or parasitism, induced resistance of the
host plant, and direct competition for space and limited resources (Janisiewicz et al., 2000).
These mechanisms bring about the desired results by reducing the infection level.
A major mechanism involved in the biological control of plant pathogens is
parasitism via degradation of the cell wall. The synthesis of extracellular hydrolases capable
of destroying fungal cell wall structural polymers is considered to be one of the possible
mechanisms.
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 36
Table 2.1. Mechanism of biocontrol (Pal et al., 2006)
The fungal cell is encapsulated by an extracellular matrix, the cell wall, which
protects it from osmotic pressure and environmental stress, and determines cell shape. The
cell wall has been described on one hand as a rigid layer of glycoproteins and
polysaccharides, and on the other hand as a dynamic structure flexible enough to cope with
cell growth. The cell wall of filamentous fungi is a complex structure mainly composed of
Type
Mechanism
Examples
Direct
antagonism
a)Hyper
parasitism/predation
Lytic/some nonlytic mycoviruses
Ampelomyces quisqualis
Lysobacter enzymogenes
Pasteuria penetrans
Trichoderma virens
Mixed-path
antagonism
a)Antibiotics
2,4-diacetylphloroglucinol,
Phenazines, Cyclic lipopeptides
b)Lytic enzymes
Chitinases, Glucanases, Proteases, lipase
c)Unregulated waste
products
Ammonia, CO2, Hydrogen cyanide
d)Physical/chemical
interference
Blockage of soil pores, Germination
signals consumption, Molecular cross-
talk confused
Indirect
antagonism
a)Competition
Exudates/leachates consumption,
Siderophore scavenging, Physical niche
occupation
b)Induction of host
resistance
Contact with fungal cell walls
Detection of pathogen-associated,
molecular patterns
Phytohormones-mediated induction
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 37
polysaccharides, chitin and glucans. It is very important in fungal morphogenesis and in
protection from diverse environmental stress. The rigid fungal cell wall determines the
shape of the fungal hyphae. It also shields the protoplast from physical and chemical stress,
such as irradiation and the toxic effects of heavy metals, and provides the fungus with a
protective outer coat, on which antigenic glycoproteins are exposed (Peberdy, 1990).
Five major components make up the cell wall of most fungi, consisting of: (1→3)-β-
glucan, (1→6) - β -glucan, (1→3)-α-glucan, chitin, and glycoproteins. Of all fungi, the cell
wall of S. cerevisiae has best been studied with regard to its structure and biosynthesis. It is
composed of (1→3) - β -glucan, that forms an alkali-soluble fraction (20% of total cell wall)
or a chitin-linked, alkali-insoluble fraction (35%), Furthermore, (1→6) - β-glucan (5%),
chitin (2%), and mannoproteins (40%) are present (Klis et al., 1997). By using electron
microscopy, it was shown that the cell-wall components are organized in a layered structure
in which (1→3)- β -glucan forms densely interwoven microfibrils present as the innermost
layer, followed by (1→6)- β -glucan and mannoproteins (Osumi, 1998). Further the
mannoproteins are attached to (1→6) - β -glucan via a glycosylphosphatidylinositol anchor.
The (1→6) - β -glucan contains (1→3) linked branches to which the reducing end of chitin
may be connected via a (1→2) or (1→4) linkage. Finally, the reducing end of the (1→6) - β
glucan is linked to a non-reducing end of (1→3)-β glucan through an as yet unknown
linkage.
The fungal cell wall though has been considered as an inert organelle, recent
analyses revealed that it is a dynamic organelle in which constituent polymers are
continuously synthesized, degraded, and chemically modified, and their structures are
rearranged (Bernard and Latgé, 2001; Popolo et al., 2001). In the model fungus Aspergillus
nidulans, chitin—a homopolymer of β-1,4-linked N-acetyl-D-glucosamine—is one of the
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 38
major cell wall components. Chitin was reported as resistance inducer against soil borne
diseases (Abd-El-Kareem et al., 2006).
If chitin in the cell wall is degraded, the solubility of the ß-glucans is affected as
well, suggesting the existence of covalent linkages between these polymers. Cross links
involving peptides (Sietsma and Wessels, 1979) and glycosidic bonds (Kóllar et al., 1995)
have indeed been found. The fibrous polysaccharides are embedded in a gel like matrix of
glucans and glycoproteins (Fig. 2.1).
Structural polysaccharides are synthesized by integral membrane proteins (Inoue et
al., 1995). UDP-N-acetylglucosamine/glucose is supplied by the cytoplasm, the synthase
catalyzes the formation of glycosidic bonds, and the polymer is extruded into the apical
wall. Inside the cell wall the polymers may be modified, e.g. deacetylated and cross linked
(Gooday, 1995). Newly synthesized chitin is highly susceptible to degradation by chitinase
as the nascent material moves away from the apex, cross linking, crystallization and the
addition of new material give the cell wall its mature, rigid properties (Vermeulen and
Wessels, 1986).
Fig. 2.1. Schematic presentation of cell wall development, showing chitin (straight
lines), ß-glucans (wavy lines) & matrix material (dots) (adapted from Gooday 1995).
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 39
Modern strategies of biological control focus increasingly on the synergism of
antibiotics and cell wall degrading enzymes in the microorganisms used for biological
control. Bacillus spp. are interesting in this context, because their large repertoire of
antibiotics (Silo Suh et al., 1994) together with several hydrolytic enzymes may be
exploited. Among cell wall degrading enzymes, chitinolytic enzyme activity has been
associated with antagonism against Basidiomycetes fungi in P.polymyxa (Mavingui and
Heulin, 1994). Bacillus sp. are also well known producers of cellulase and hemicellulase
(xylanase and mannanase) activities which could be involved in degradation of cellulose,
galactomannan or mannoprotein-containing cell walls of Oomycetes (Bartnicki-Garcia,
1987; Kim and Kim, 1993). Many microorganisms have been reported as good sources for
the production of cellulases and hemicellulases. The soft rot fungus Trichoderma reesei has
been studied in detail due to its ability to secrete large amounts of enzymes (up to 35 g per
liter) (Wakayama, 1984).
Fungal cell walls are composed of several polysaccharides. Therefore, it is likely
that lysis of these cell walls is caused by concerted action of various enzymes that degrade
different polysaccharides. Among the mycolytic enzymes, chitinases (EC 3.2.1.14) and β-
1,3-glucanases (EC 3.2.1.6; 3.2.1.39), which are quite widespread among the representatives
of saprophytic soil microflora, attract the most attention. However, the active involvement
of enzymes in inhibiting the growth of phytopathogenic fungi has been confirmed only for
individual well-studied groups of microbial antagonists (Markovich and Kononova, 2003).
The role of extracellular hydrolases of most saprophytic antagonistic bacteria in the
biological control of phytopathogens is poorly studied, since their main function is supposed
to be the decomposition of organic matter from the dead fungal mycelium for nutrient
extraction (Chernin, 2001). Cell-wall degrading enzymes such as cellulases and glucanases
are especially important in the breakdown of the cell wall of oomycete pathogens such as
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 40
Pythium spp. and Phytophthora spp. Cellulases and glucanases are produced by several
fungi, bacteria, Streptomycete Actinomycetes and non Streptomycete Actinomycetes (Khaled
and Tarabily, 2006).
Chitinase, glucanase and other hydrolytic enzymes have many roles in wide range of
different biological systems. These enzymes are usually extracellular, of low molecular
weight and high stability. In addition they may be produced in multiple forms or isozymes
that differ in charge, size, regulation, stability and ability to degrade cell walls (Koga et al.,
1988). Pathogens as predators of chitinous organisms produce chitinases where as hosts to
chitinous pathogens, including plants and humans produce chitinase to protect themselves
(Gooday, 1999). The involvement of chitinase and other cell wall degrading enzymes and
their genes in penetration, pathogen ramification, plant defence induction and symptom
expression has been studied extensively, however conclusive evidence for or against a role
of any particular enzymes activity in any aspect of pathogenesis has been difficult to
discern (Walton, 1994).
Figure 2.2 Molecular structure of chitin.
Chitin, the unbranched homopolymer of N-acetyl glucosamine in a β-1, 4 linkage is
a structural component of cell walls in most of the fungi (Fig.2.2) (other than Oomycetes).
Chitinases which hydrolyse this polymer are produced by various organisms and have been
implicated in the biocontrol process (Mitchell and Alexander, 1962; Henis and Chet, 1975).
The antifungal activity of chitinases is due to its capacity of degrading chitin to its
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 41
oligomers (chito oligosaccharides) and/or monomers (N-acetylglucosamine). These
enzymes are effective tools for complete degradation of mycelia and conidial walls of
phytopathogenic fungi (de la Cruz et al., 1995a).
Chitin can be degraded via either of the two main pathways. In one, degradation is
initiated by the chitinase induced hydrolysis of the β-1, 4-glycosidic linkage, a process
termed the chitinolytic mechanism. Alternatively, the polymer is first deacetylated and
thereafter hydrolyzed by chitosanases. Chitinases (EC 3.2.1.14) are glycosyl hydrolases and
are extensively distributed among plants, fungi, bacteria and viruses. In higher plants
chitinases are used as defence against plant pathogens (Koga et al., 1988). The enzyme has
a mixture of secondary structures, including 10 α-helical segments and one three-stranded β-
sheet. These enzymes are found in low levels in healthy plants; however their expression
increases during pathogen attack. The production of chitinases elicits other plant responses
including the synthesis of antifungal phytoalexins (Gooday, 1999).
Another enzymatic system that is involved in cell wall degradation by an
antagonistic organism is β -glucan degrading enzymes. Cellulolytic enzymes could provide
a means for degrading fungal mycelia and yeast cells to use them as biomass resources or to
control pathogenic fungi. Many microorganisms are known to produce simultaneously
plural enzymes that hydrolyze in concert a particular insoluble polysaccharide. Multiple, β-
1,3-glucanase systems are present in many microorganisms (Fiske et al., 1990). The
multiple systems seem to contain not only genetically different isozymes but also small
products that result from each isozyme by proteolytic processing. Two mechanisms of
glucan degradation have been reported: exo- and endoglucanases, both of which act
synergistically in glucan degradation. β - glucan degrading enzymes are classified according
to the type of β -glucosidic linkages: 1, 4- β glucanases (including cellulases), 1, 3- β -
glucanases, and 1,6- β -glucanases (Pitson et al., 1993). As 1, 3-β-glucan is a structural
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 42
component of fungal cell walls, the production of extracellular 1,3-β-glucanases has been
reported as an important enzymatic activity in biocontrol microorganisms. In addition to
chitin and glucans, filamentous fungi cell walls contain proteins. Thus, the production of
proteases may play a role in antagonism (Sivan and Chet, 1989; Flores et al., 1997).
β-Glucanases, produced by several fungi and bacteria are one of the most potent
enzymes for degrading fungal cell walls (Chet, 1998). More recently, intensive efforts have
been made to use the biocontrol agents for protecting fruit and vegetable crops from post-
harvest diseases (Batta, 2004). Antagonists were selected following evidence of direct
interactions or after the demonstration that substances or antibiotics toxic to the potential
pathogens were secreted into the growth medium.
β-1,3 glucanase, hydrolyze the O- glycosidic linkages of β-glucan chains by two
mechanisms, the 1st is where, exo -β-1,3 glucanase (EC 3.2.1.58) hydrolyzes a substrate by
sequentially cleaving glucose residues from the non reducing end , and in the 2nd
, endo-β-
1,3 glucanase (EC 3.2.1.39) cleaves β-linkages at random sites releasing oligosaccharides
(Moataza, 2006).
Cellulases are responsible for the hydrolysis of the β-1, 4-glucosidic bonds in
cellulose. Ordinarily it was accepted that effective biological hydrolysis of cellulose to
glucose requires synergistic collaboration of three different kinds of enzymes: endo-β-1, 4-
glucanase (EC 3.2.1.4, EG) which randomly cleaves internal linkages in cellulose chains;
cellobiohydrolase (EC 3.2.1.91, CBH) which specifically cleaves cellobiosyl units from
non-reducing ends of cellulose chains; and β-D-glucosidase (EC 3.2.1.21) which cleaves
glucosyl units from cellooligo-saccharides converting cellobiose into glucose and thus
completing the cellulolysis (Jabber, 2004; Schulein, 2000).
Montealegre et al. (2003) evaluated the mechanism of antagonism of B.subtilis and
B.lentimorbis against R.solani infection in tomato. The mechanism used by these bacteria
Isolation and characterisation of Bacillus sp. mycolytic enzymes for plant defense............
Ashwini N. Ph.D. Thesis Department of Microbiology, Jain University, Bangalore. Page 43
was the secretion of volatile and difusible metabolites but not of fungal cell wall hydrolytic
enzymes.
Kavitha et al. (2005) have reported the application of PGPR isolates (Bacillus sp.
and Pseudomonas sp.) for the control of damping off caused by Pythium aphanidermatum.
In spite of its direct action these triggered the defense related enzymes involved in phenyl
propanoid pathway and phenolics.
Vivekananthan et al. (2004) evaluated the lytic enzyme induced biocontrol of mango
anthracnose by talc based formulation of Pseudomonas flourescens, Bacillus subtilis and
Saccharomyces cerevisiae under endemic conditions. Plant growth promoting factors and
defence mediating enzymes produced by the biocontrol agents were found to collectively
contribute to suppressing the pathogen and increasing yield.
Moataza Saad (2006) has reported the lytic enzyme mediated biocontrol of R. solani
and P. capsici by Pseudomonas fluorescences NRC1 and Pseudomonas fluorescences
NRC3. The agents had a relatively strong lytic activities of chitinase, β-1, 3 and β -
1,4glucanases, protease and lipase , toward the tested fungi.
Minggou Zhou et al. (2007) isolated an antifungal protein, Bacisubin, from B.subtilis
which exhibited inhibitory activity on mycelial growth in Magnaporthe grisease, Sclerotinia
sclerotiorum, Rhizoctonia solani, Alternaria oleracea, A. brassicae and Botrytis cinerea.
Virgilio Mojica- Marin et al. (2008) have reported antibiosis mediated antagonism of
R.solani of chilli by Bacillus thuringiensis. In the volatile antibiotics assay, the strains GM-
11 and GM-121 showed the best inhibitory effect over R. solani growth.
Ko et al. (2009) worked with several rhizobacteria and isolated a strain of
Lysobacter antibioticus HS124 and demonstrated its antifungal activity against various
pathogens including Phytophthora capsici, a destructive pathogen of pepper plants. L.
antibioticus HS124 was capable of producing lytic enzymes such as chitinase, beta-1,3-
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glucanase, lipase, protease, and an antibiotic compound as various factors contributing to
antagonism.
2.3 Bacillus as Biological Control Agents:
The rhizosphere is a zone around plant roots where microbes interact and inter- and
intra-species interactions of microbes, such as bacteria, fungi and protozoa, occur due to the
presence of a rich and diverse microbial food source (Bais et al., 2006). Among the
interactions between plants and microbes, the role of rhizosphere bacteria (rhizobacteria)
has been of great interest in efforts to stimulate plant growth, as some rhizobacteria, referred
to as plant growth-promoting rhizobacteria (PGPR), have been shown to significantly
increase crop yield in the greenhouses and fields (Kloepper et al., 2004). By comparison,
Bacillus spp. and Paenibacillus spp. are considered less potent PGPR strains than Gram-
negative bacteria, because bacilli typically have longer generation times and are isolated at
lower population densities from plant roots than Pseudomonas spp. (Bakker et al., 2007).
However, interest in endospore-forming bacilli has been revived recently in light of
commercialization efforts with fluorescent pseudomonads, which revealed in early trials that
biocontrol and biofertilizer products based on Pseudomonas spp. fail due to an insufficient
shelf life (Kloepper et al., 2004). The ability of many strains of Bacillus to colonize the
rhizosphere of cultivated plants and stimulate their growth is of great importance in this
respect. Aktuganov et al. (2007) worked with Bacillus sp. 739, which is used as a biological
basis for the experimental preparation Batsispetsin BM, effective against fungal pathogenic
agents of cereal diseases. The efficacy of this strain as a biocontrol agent was determined
not only by its antagonistic activity but, probably also by the production of substances of a
phytohormonal nature.
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Dal Soo Kim et al. (1997) explored the Strain L324-92 as a novel Bacillus sp. and
confirmed its biological activity against three root diseases of wheat, namely take-all caused
by Gaeumannomyces graminis var. tritici, Rhizoctonia root rot caused by Rhizoctonia
solani AG8, and Pythium root rot caused mainly by Pythium irregulare and P. ultimum, and
grows at temperatures from 4 to 40°C.
Kavitha et al. (2005) worked on several isolates of PGPR belonging to the species of
Bacillus subtilis and Pseudomonas sp. and screened them for their effectiveness in
controlling damping off of chilli (Capsicum annuum L). Among the different isolates,
Bacillus subtilis (CBE4) and Pseudomonas chlororaphis (BCA+) proved to be effective
against Pythium aphanidermatum under in vitro.
The work of Virgilio Mojica-Marín et al. (2008) suggests that out of the 60 strains of
Bacillus thuringiensis checked for antagonistic effectiveness against Rhizoctonia solani
causing damping-off and root and stem rot, 16 displayed inhibitory effect. These results
suggested that the B.thuringiensis strains studied have an excellent potential to be used as
biocontrol agents of R.solani in chilli pepper.
Mirik et al. (2008) isolated 3 Bacillus strains from soil samples of the rhizospheres
of peppers grown in greenhouses and fields to suppress the size of the population of X.
axonopodis pv. vesicatoria. Results indicated that disease development decreased by 11%-
62% and 38%-67% in pepper plants inoculated with the 3 Bacillus strains alone and in
combination, respectively, in greenhouse and field experiments.
The possibility to reduce Phytophthora blight of Chilli using phosphate-solubilizing
bacteria was investigated by Akgül and Mirik, (2008) in growth room and field experiments.
The pepper plants, inoculated with the pathogen after pre-inoculation with three phosphate-
solubilizing strains of Bacillus megaterium employed alone or in combination, were
monitored for growth parameters and disease severity. Inoculation with the selected strains
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significantly reduced disease severity in field experiments and two strains increased the
yield by 36.2 and 47.7% compared to untreated controls.
Beatriz et al. (2009) determined the in vitro activity of 13 native strains of Bacillus
sp. isolated from soil against Macrophomina pasheolina using a dual assay in nutrient agar.
All the strains showed inhibition of radial growth from 31-80%.
Hamdia Ali and Kalaivani Nadarajah (2012) determined the effectiveness of
B.subtilis isolates as a biological control agent against Rhizoctonia solani. B. subtilis had
excellent suppression of pre (8.67, 8.33, 13, 8.67, 8.67 and 8.67%) and post (9, 8.67, 9.33,
14, 9 and 14%) emergence of disease in R. solani inoculated soil.
2.3.1 Antagonistic Mechanism of Bacillus
The extracellular cell wall degrading enzymes excreted by many strains of Bacillus
are traditionally included in the concept of mycoparasitism, due to their integral role in
direct physical interactions (Abdullah et al., 2008). In addition to secretion of proteases, the
ability to degrade polysaccharides is a common property. Various types of secreted amylase
have been characterized from mesophilic (e.g. Bacillus amyloliquefaciens, Bacillus
licheniformis) and alkaliphilic species (Vihinen and Mantsala, 1989). Other polysaccharide-
degrading enzymes secreted by Bacillus species include different types of cellulases (Blanco
et al., 1998), xylanases (Blanco et al., 1999) and lichenases (1, 3- 1, 4-β-glucanases)
(Sanchez-Torres et al., 1996). In contrast to the above polysaccharides, only a few Bacillus
species are known to hydrolyse the second most abundant polysaccharide in nature, chitin,
and its deacetylated derivative chitosan B. amyloliquefaciens, Bacillus megaterium and
Bacillus subtilis are counted among the degraders of shrimp shell waste (Sabry, 1992),
although their detailed degradation properties have been rarely investigated (Frandberg &
Schnuer, 1998). Chitinases have been identified within Bacillus cereus (Pleban et al., 1997;
Trachuk et al., 1996), Bacillus circulans (Watanabe et al., 1992) and Bacillus thuringiensis
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(Sampson & Gooday, 1998). Much is known about B. circulans chitinases and their
corresponding genes (Wiwat et al., 1999). Chitosanases have been characterized from B.
circulans (Saito et al., 1999), B. cereus (Kurakake et al., 2000) and some unidentified
Bacillus species (Izume et al., 1992). Chitinases are reported to play a protective role
against fungal pathogens. Besides its ability to attack the fungal cell wall directly, chitinases
release oligo-N-acetyl glucosamines that function as elicitors for the activation of defense-
related responses in plant cells (Ren and West, 1992).
Chitinases have been isolated from variety of bacteria including Bacillus spp. and
some of them are reported to produce multiple forms of chitinases with different molecular
masses (Vaidya et al., 2001; Wen et al., 2002; Someya et al., 2003; Woo and Park, 2003;
Dahiya et al., 2005; Ajit et al., 2006). Chitinase production was reported in different species
of Bacillus such as Bacillus amyl-oliquefaciens (Sabry, 1992), Bacillus cereus (Chang et al.,
2007), Bacillus circulans (Chen et al., 2004), Bacillus licheniformis (Waldeck et al., 2006),
Bacillus megaterium (Sabry, 1992), Bacillus pabuli (Frandberg and Schnurer, 1994),
Bacillus stearothermophilus (Sakai et al., 1994), Bacillus subtilis (Wang et al., 2006),
Bacillus thuringiensis sub sp. Aizawai (de la Vega et al., 2006), B. thuringiensis sub sp.
Kurstaki (Driss et al., 2005).
Enzymes capable of hydrolyzing (1, 3)-β, and (1, 6)- β-glucans are frequently
synthesized by fungi and bacteria ( Chesters and Bull,1963). Within the genus Bacillus, β-
glucanases have been described from B. circulans WL-12 , B. polymyxa several strains of
B. subtilis (Borriss, 1976 ), and an alkalophilic isolate (Horikloshi and Atsukawa 1973).
These enzymes are believed to be of considerable ecological significance (Mann et al.,
1978). Strains of Bacillus subtilis also have been studied as biocontrol agents of plant
pathogens.
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Diverse microorganisms secrete and excrete other metabolites that can interfere with
pathogen growth and/or activities. Many microorganisms produce and release lytic enzymes
that can hydrolyze a wide variety of polymeric compounds, including chitin, proteins,
cellulose, hemicellulose, and DNA. Expression and secretion of these enzymes by different
microbes can sometimes result in the suppression of plant pathogen activities directly. For
example, control of Sclerotium rolfsii by Serratia marcescens appeared to be mediated by
chitinase expression (Ordentlich et al., 1988) and β-1,3-glucanase contributes significantly
to biocontrol activities of Lysobacter enzymogenes strain C3 (Palumbo et al., 2005). While
they may stress and/or lyse cell walls of living organisms, these enzymes generally act to
decompose plant residues and nonliving organic matter.
Nielsen et al. (1996) isolated glucanolytic Bacillus sp. from barley rhizosphere soil,
16 out of 100 isolates exhibited mycolytic enzyme mediated antagonism of plant pathogenic
microfungi. The antagonistic isolates were identified as Paenibacillus (Bacillus) polymyxa
(2 strains) and Bacillus pumilus (13 strains).
Pleban et al. (1997) worked on Bacillus cereus strain 65, previously isolated as an
endophyte of Sinapis, showed to produce and excrete a chitinase with an apparent molecular
mass of 36 kDa. Application of B. cereus 65 directly to soil significantly protected cotton
seedlings from root rot disease caused by Rhizoctonia solani.
Helisto et al. (2001) worked with Bacillus sp. X-b, a biocontrol agent against certain
plant pathogenic fungi. The strain secreted a complex of hydrolytic enzymes, composed of
chitinase, chitosanase, laminarinase, lipase and protease. Homogenized mycelium of
basidiomycete Macrolepiota procera induced activities of these enzymes more effectively
than colloidal chitin or partially purified cell walls of another basidiomycete Polyporus
squamosus.
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Basha and Kandasamy Ulaganathan (2002) worked on a soil bacterium, Bacillus sp.
strain BC121, isolated from the rhizosphere of sorghum which showed high antagonistic
activity against Curvularia lunata. In dual cultures, the Bacillus strain BC121 inhibited the
C. lunata up to 60% in terms of dry weight. This strain also produced a clear halo region on
chitin agar medium plates containing 0.5% colloidal chitin, indicating that it excretes
chitinase.
Aktuganov et al. (2003) tested 70 Bacillus spp. strains antagonistic to
phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid
media with 0.5% colloidal chitin. The chitinolytic activity of almost all of these 19 strains
grown in liquid cultures ranged from 0.1 to 0.3 U/ml. One of the 19 strains exhibited
exochitinase activity. In addition to chitinase, two strains also produced chitosanase and one
strain, produced β-1, 3- glucanase.
Viswanathan et al. (2003) tested several strains for chitinolytic activity against
Colletotrichum falcatum from sugarcane rhizosphere, 12 strains showed a clearing zone on
chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7
and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger
than 10 mm. They showed increased levels of activity of N-acetylglucosaminidase and β-
1,3-glucanase when grown on minimal medium containing chitin or cell wall of the
pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T.
harzianum T5 inhibited conidial germination and mycelial growth of the pathogen.
Reyes-Ramirez et al. (2004) used Bacillus thuringiensis var israelensis to produce
chitinase on shrimp wastes by fermentation at 30 °C and 250 rpm for 120 h. The enzyme
was concentrated by ultrafiltration and was adjusted to pH 5.8. Antifungal chitinase activity
on phytopathogenic fungi was investigated in growing cultures and on soybean seeds
infested with Sclerotium rolfsii. Fungal inhibition was found to be 100% for S. rolfsii; 55%
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to 82% for A. terreus, A. flavus, Nigrospora sp, Rhizopus sp, A. niger, Fusarium sp, A.
candidus, absidia sp, and Helminthosporium sp; 45% for Curvularia sp; and 10% for
A. fumigatus (P < 0.05). When soybean seeds were infected with S. rolfsii, germination was
reduced from 93% to 25%; the addition of chitinase (0.8 U/mg protein) increased
germination to 90%.
Huang et al. (2005) worked with Bacillus cereus 28-9, a chitinolytic bacterium
isolated from lily plant in Taiwan. This bacterium exhibited biocontrol potential on Botrytis
leaf blight of lily as demonstrated by a detached leaf assay and dual culture assay. The
organism was able to excrete atleast two chitinases (ChiCW and ChiCH). An in vitro assay
showed that the purified ChiCW had inhibitory activity on conidial germination of Botrytis
elliptica, a major fungal pathogen of lily leaf blight.
According to the work of Aktuganov et al. (2007) the mycolytic bacterial strain
Bacillus sp. 739 produced extracellular enzymes which degrade in vitro the cell walls of a
number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated
with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process
correlated with the levels of the β-1,3-glucanase and protease activity. Among the enzymes
of this complex, chitinases and β-1,3-glucanases hydrolyzed most actively the disintegrated
cell walls of B. sorokiniana. . However, only β-1, 3-glucanases were able to degrade the cell
walls of native fungal mycelium in the absence of other hydrolases, which is indicative of
their key role in the mycolytic activity of Bacillus sp. 739.
Kamil et al. (2007) screened four hundred bacterial isolates from rhizosphere of
some plants collected from Egypt for production of chitinase enzyme. The most potent
chitinolytic bacterial species were identified as Bacillus licheniformis, Stenotrophomonas
maltophilia, Bacillus licheniformis and B. thuringiensis. In vitro MS1 and MS3 were the
most active species, so they suppressed the growth of all tested pathogenic fungi
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(Rhizoctonia solani, Macrophomina phasiolina, Fusarium culmorum, Pythium sp,
Alternaria alternata and Sclerotium rolfsii).
Shanmugaiah et al. (2008), isolated a total of 39 chitinolytic bacteria from 77
rhizosphere soil samples collected from different crop fields in Tamil Nadu state, India.
Among them, a strain designated as MML2270, produced highest chitinolytic activity in
primary and secondary screening in colloidal chitin agar, was selected and later identified as
Bacillus laterosporous.
Beatriz et al. (2009) determined the in vitro activity of 13 native strains of Bacillus
sp. isolated from soil against Macrophomina pasheolina using a dual assay in nutrient agar.
The crude extract of the strain of chitinases LUMB0 04 inhibited the growth of M.
phaseolina by 30%. The strains of chitinolytic Bacillus sp. isolated from soils showed
antifungal activity and have the potential to be used in biological control of M. phaseolina.
The work of Suryanto et al. (2010) was the evaluation of the ability of chitinolytic
bacteria to suppress Fusarium wilt of red chilli disease by soaking red chilli seeds in the
bacterial isolates solution for 30 minutes prior seedling. Percentage of seedling of treated
chilli seed at end of study (4-weeks) ranged from 46 to 82.14%.
The aim of the investigation by Praveen Kumar et al. (2012) was to study the
hydrolytic enzymes viz., chitinase, protease, β-1, 3 glucanase and cellulase from the isolates
of Bacillus sp. (twenty eight) which were isolated from tomato rhizospheric soil in IIVR
farm (DPNSB-1 to 7), IIHR farm (DPNSB-8 to 15), IARI farm (DPNSB-16 to 20) and farm
of APHU (DPNSB-21 to 28). Among the strains, IARI isolate of DPNSB-18 exhibited the
highest chitinase activity (4.65 IU/ml), IIHR isolate of DPNSB-15 produce highest protease
activity (0.79 IU/ml), maximum , β-1, 3 glucanase production was noted in Bacillus strains
viz., DPNSB-14 (IIHR isolate), DPNSB-2 (IIVR isolate) and DPNSB-20 (IARI isolate),
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ranged from 0.24 IU/ml to 0.39 IU/ml, cellulose production was made by isolates of IIVR,
DPNSB-3 (0.75 IU/ml) and DPNSB-1 (0.60 IU.ml) respectively.
2.3.2 Isolation and screening
Chitinases are important enzymes in biotechnology and bioprocessing. Screening
and isolation of chitinolytic microorganisms is usually performed on chitin agar plates and
then chitinase activities are assayed by determination of reducing end sugar. Chitinase
activity can be qualitatively assayed by determining the clearance zone developed around
the colonies growing on the colloidal chitin agar medium (Cody, 1989; Wirth and Wolf,
1990). The potency of the isolates for chitinase production is determined on the basis of
ratio of zone of clearance (CZ) to colony size (CS) (Cody, 1989).
Degradation of β-glucan by fungi is often accomplished by the synergistic action of
both endo and exo β-glucanases; in fact, in most cases multiple β-glucanases rather than a
single enzyme have been found. A number of fungal β-1,3-glucanases have been the
subject of basic and applied research, as they seem to have different functions during
development and differentiation (Peberdy, 1990). It has been suggested that β-1,3-
glucanases play a nutritional role in saprophytes and mycoparasites (Chet, 1987; Sivan and
Chet, 1989), and these enzymes have also been implicated in autolysis (Stahmann et al.,
1993). Furthermore, β-1,3-glucanases are among the plant defense responses to pathogen
attack (Simmons, 1994).
Trinitrophenyl-CMC (TNP-CMC) was chosen as the substrate for screening of β 1,4
cellulase producers, in the hope that low amounts of cellulolytic activity would be detected.
Agar plates containing CMC or cellulose were used to screen for cellulolytic Bacillus
species, and clearing zones were observed around isolated Bacillus colonies (Robson,
1984).
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2.4 Induction and Optimisation of lytic enzymes production by B.subtilis
Before formulation of mycolytic microorganisms, the favourable medium
constituents are required for increasing the growth and production of chitinolytic enzymes.
Moreover to attain a cost effective process, it is imperative to select a growth medium.
There are large numbers of reports available on conventional or one-factor-at a time and
statistical method for designing/selecting the media for enhancing the growth and
production of chitinolytic enzymes (Vaidya et al., 2001; Felse and Panda, 1999; Madhavan-
Nampoothiri et al., 2004). However, the conventional or one-factor-at a time approach
becomes extremely time consuming, expensive and unmanageable when large numbers of
variables have to be studied and does not depict the combined effect of all the factors
involved.
Moreover, the method requires large number of experiments to determine optimum
levels, which are unreliable (Halland, 1989; Vaidya et al., 2001). Optimizing all the
affecting parameters by statistical experimental designs can eliminate these limitations of a
single factor optimization process collectively (Halland, 1989; Montogomery, 2000). The
statistical methodologies are preferred because of various advantages in their use such as
rapid and reliable short-listing of nutrients, understanding the effect of the nutrients at
varying concentrations and significant reduction in total number of experiments resulting in
saving time, glassware, chemicals and manpower (Srinivas et al., 1994; Carvalho et al.,
1997; Vaidya et al., 2003). There are many other techniques available for screening and
optimization of process parameters including non-statistical and self optimization
techniques (Felse and Panda, 1999).
However, before statistical optimization of medium for production of desired
product from a new source bacterium it is essential to screen large number of possible
medium constituents. Component replacing is the most commonly used method for
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screening number of carbon, nitrogen and phosphorous sources (Jatinder et al., 2006) while
the effect of surfactants, metal ion, antibiotics etc is checked by one factor at time approach
(Patidar et al., 2005). This approach can generate information on medium constituents for
desired product from organism under study and can also identify new components affecting
its production.
Microbial degrading enzymes of the cell wall of fungal pathogens have been
reported (Lorito et al., 1998). The production of extracellular β -1,3 and β -1,4 glucanases,
chitinase, lipase and protease increased significantly when Pseudomonas species was grown
in a medium supplemented with either autoclaved mycelium or host fungal cell walls. These
observation, together with the fact that chitin β-1,3-glucan and protein are the main
structural components of most pathogenic fungal cell walls (Ziedan et al., 2005) suggested
that lytic enzymes produced by some microorganisms play an important role in the
destruction of plant pathogens. It has been reported that production of β-1, 3-glucanases by
T.harzianum is dependent on the carbon source available (de La Cruz et al., 1993). The
production of extracellular glucanases in microorganisms is significantly influenced by a
number of factors such as temperature, pH, aeration and medium constituents. The
relationship between these variables has a marked effect on the ultimate production of these
enzymes and thus lytic enzyme mediated antibiosis (Immanuel et al., 2006).
He-Guo et al. (2002) investigated the cultural conditions for β Glucanase production
by Bacillus subtilis ZJF- 1 A5. Temperature had a great effect on β Glucanase production
which maximised at optimal temperature of 37°C and decreased significantly at higher
temperatures. Age, size of inocula and shaking speed were identified as the key factors
affecting the production of the enzyme.
Manjula and Podile (2005) conducted the study to describe the optimum conditions
required for the production of β-1, 4-N-acetyl glucosaminidase (NAGase) and β -1,3-
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glucanase by a biocontrol strain of Bacillus subtilis AF 1. The isolate was grown in minimal
medium with colloidal chitin (3.0%) and yeast extract (0.3% YE ) and incubated at pH 7.0
and 30°C on constant shaker at 180 rpm for 6 days produced highest amounts of NAGase.
Presence of 0.5 mM of phenyl methyl sulfonyl fluoride (PMSF) and 0.04% of Tween 20
further improved the enzyme production. B. subtilis AF 1 grown in minimal medium with
laminarin (1%) and yeast extract (0.3%) for 3 days produced maximum amount of beta-1,3-
glucanase.
Chhatpar et al. (2009) statistically optimised the medium components for improved
chitinase production by Paenibacillus sp. D1. Urea, K2HPO4, chitin and yeast extract were
identified as significant components influencing chitinase production using Plackett–
Burman method. Response surface methodology (central composite design) was applied for
further optimization. The concentrations of medium components for improved chitinase
production were as follows (g l-1
): urea, 0.33; K2HPO4, 1.17; MgSO4, 0.3; yeast extract,
0.65 and chitin, 3.75. This statistical optimization approach led to the production of 93.2 ±
0.58 U ml-1
of chitinase.
Shanmugaiah et al. (2008) optimised the production of chitinase by B. laterosporous
using different growth media, substrate concentrations, pH, temperature and incubation
period. The maximum chitinase production was observed in yeast nitrogen based medium
(YNB) amended with 0.3% colloidal chitin at pH 8.0 and 35°C after four days of
inoculation. Under this optimized growth condition, B. laterosporous MML2270 produced a
total chitinase activity of 59.05 units/ml on the fifth day as against only 19.7 units/ml in the
initial YNB medium stage, which was a three-fold increase.
Natarajan and Murthy, (2010) analyzed the fermentative physicochemical
parameters such as agitation speed, temperature and pH by classical approach method for
the production of chitinase enzyme and growth of bacterial strain of Serratia marcescens.
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The parameters set up for reaching maximum response for production of chitinase and cell
growth were obtained at 250 rpm agitation speed, 30 ºC temperature and the pH of 9. The
chitinase production increased three fold using optimized culture compositions and culture
conditions (44.01 U/mL).
Deepmoni et al. (2011) were able to enhance cellulase activity of Bacillus subtilis
AS3 by optimizing the medium composition by statistical methods. The enzyme activity
with unoptimised medium with carboxymethylcellulose (CMC) was 0.07U/mL and that was
significantly enhanced by CMC, peptone, and yeast extract using Placket-Burman design.
The combined effects of these nutrients on cellulose activity were studied using 22 full
factorial central composite designs. The optimal levels of medium components determined
were CMC (1.8%), peptone (0.8%), and yeast extract (0.479%). The maximum enzyme
activity predicted by the model was 0.49U/mL which was in good agreement with the
experimental value 0.43U/mL showing 6-fold increase as compared to unoptimised
medium. The enzyme showed multisubstrate specificity, showing significantly higher
activity with lichenan and β-glucan and lower activity with laminarin,
hydroxyethylcellulose, and steam exploded bagasse. The optimised medium with lichenan
or β-glucan showed 2.5- or 2.8-fold higher activity, respectively, at same concentration as of
CMC.
Prasad Loni and Shyam Bajekal (2011) isolated a potent chitinolytic bacteria from
alkaline –saline environment which was identified as Bacillus firmus. The chitinase enzyme
obtained from this bacterium was found to have maximum activity at alkaline pH range and
most stability at pH-10. The enzyme had maximum activity at temperature of 37°C and 3%
salt concentration.
Kavikarunya et al. (2011) optimized an industrial enzyme chitinase produced by
Bacillus subtilis. The enzyme was purified and its antifungal activity was investigated
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against plant pathogens. The isolate showed highest chitinolytic activity in colloidal chitin
agar that degraded chitin with 0.6 mm zone of clearance. The production of chitinase by
Bacillus subtilis was optimized under different media, substrates, substrate concentrations,
pH, temperature and incubation period. The maximum chitinase production was observed in
Luria Bertani Broth amended with 0.3% colloidal chitin at pH 7.0 and temperature 35˚C
after four days of incubation. The enzyme was partially purified by Dialysis method. Protein
concentration of 200μg/ml was estimated according to Lowry’s method. The Chitinase had
antifungal activity against plant pathogens viz, Aspergillus niger, Aspergillus flavus and
Penicillium chrysogenum.
Mehdi Fazelia et al. (2011) employed a two step approach for the optimisation of
culture conditions for chitinase production by Bacillus pumilus. First, the effects of several
medium components were studied using the Plackett-Burman design. Among various
components tested, chitin and yeast extract showed positive effect on enzyme production
while MgSO4 and FeSO4 had negative effect. The linear model proved to be insufficient for
determining the optimum levels for these components due to a highly significant curvature
effect. In the second step, Box-Behnken response surface methodology was used to
determine the optimum values. The optimum concentrations for chitin, yeast extract,
MgSO4 and FeSO4 were found to be 4.76, 0.439, 0.0055 and 0.019 g/L, respectively, with a
predicted value of chitinase production of 97.67 U/100 ml. Using this statistically optimized
medium, the practical chitinase production reached 96.1 U/100 mL.
Solanki et al. (2012) isolated four antagonistic bacteria namely, Bacillus megaterium
MB3, B. subtilis MB14, B. subtilis MB99 and B. amyloliquefaciens MB101 which were
able to produce chitinase, β-1, 3-glucanase and protease in different range in the presence of
Rhizoctonia solani cell wall as a carbon source.
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2.5 Assessment of the role of mycolytic enzymes in antagonism by mutagenesis
approach
Biocontrol agents of plant diseases are a particularly interesting and under-
investigated source of biologically active compounds, because antibiosis is a frequent
mechanism by which they exert their antagonistic activities. As a promising biocontrol
agent, P. flocculosa has been studied extensively with regards to its mode of action. A
number of microscopical and chemical studies have suggested that the principal mode of
action of P. flocculosa is antibiosis and that production of fatty acids by the fungus plays an
important role in its biocontrol potential (Avis et al., 2001). For this role to be confirmed,
the creation of mutants with diminished or no antagonistic properties would be invaluable.
Identifying the different mechanisms of biocontrol is important because it may
provide a means of attacking pathogens with a broader spectrum of microbial weapons.
Although naturally occurring organisms provide a major source of mycolytic enzymes,
genetic improvement plays an important role not only in ascertaining their role in biocontrol
but also in other biotechnological applications. Mechanism of antibiosis can be further
proved by establishing the sensitivity of the culture filtrate of the biocontrol agent to heat
and proteinases.
Classical mutagenesis with physical and/or chemical agents followed by titre test of
a large number of isolates has been used successfully to improve the productivity of several
fungal metabolites and enzymes (Bai et al., 2004; Pandey et al., 2000; Rubinder et al.,
2000).
Genetic and biochemical investigation in bacteriology are often initiated by the
isolation of mutants. The power of mutational analysis derives from the ability to query an
organism incisively. Even following exposure to mutagens mutations in a particular gene
might occur, at most, with an incidence of 10. In the absence of mutagens, the incidence of
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spontaneous mutation might be only 10-10
. Thus, mutant isolation requires only the use of
selection and /or screening techniques that can reveal or identify the rare colonies arising
from mutant cells (Lee et al., 2003).
There are number of different methods available for strain improvement or raising
mutants for the assessment of lytic enzyme production, the predominant being random
mutagenesis through physical (UV, Gamma etc.) and chemical (EMS) agents which have
been employed to obtain improved biological strains, including Pantoea dispersa (Gohel et
al., 2004) and Alcaligenes xylosoxydans (Vaidya et al., 2003).
A). Selection:
Selection strategies use conditions that permit growth of only the desired mutant.
Selection techniques are powerful because rare mutants can be isolated from a large
background of non mutants in a single step. When there are several different solutions to the
selective challenge imposed on the microbial population, more than one kind of mutant can
be isolated.
B). Screening:
Many mutations of interest cannot be selected directly. For such mutations one has
to use screens that discriminate between wild-type and mutant phenotypes. Screening
strategies use tests that are often applied to distinguish wild type from mutant phenotypes.
Screens can be designed to be highly specific at the biochemical level and are especially
useful when there is no obvious selection for the specific phenotype.
UV-Induced Mutants:
Two factors that can interfere with efficient UV mutagenesis are the presence of
UV- absorbing nutrients (aromatic compounds such as tryptophan or nucleic acid bases) and
the shielding of cells by high cell densities. An additional interfering factor is photo-
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reactivation. Many bacteria are able to repair some kinds of UV- induced DNA damage
(pyrimidine dimers) in a visible-light-dependent reaction.
Ethyl methanesulfonate-Induced Mutants:
Other alkylating agents that are potent bacterial mutagens include ethyl
methanesulfonate, diethyl sulfate, and methyl methanesulfonate (MMS). EMS ethylates
guanine at the O-6 position, thereby inducing primarily G:C-A:T transition mutations. In E.
coli, EMS mutagenesis is not dependent on specific DNA repair activities whereas MMS
mutagenesis is largely SOS dependent. EMS may therefore be a more tractable mutagen for
organisms not known to be capable of carrying out SOS mutagenesis.
Basha and Ulaganathan (2002) studied the role of the Bacillus strain BC121 in
suppressing the fungal growth in vitro in comparison with a mutant of that strain, which
lacked both antagonistic activity and chitinolytic activity. The bacterial culture was
incubated in 5 ml of nitrosoguanidine solution (1 mg/ml NTG) suspended in 10 mM Tris
maleic acid (pH 6.0). The colonies were screened for the loss of antifungal activity. Out of
550 putative mutant colonies tested, one showed no antagonistic property against C.lunata.
The extra-cellular protein precipitate from Bacillus strain BC121 culture filtrate had
significant growth retarding effect and mycolytic activity on C. lunata. The protein extract
from the wild strain, when tested on SDS–PAGE gel showed a unique band corresponding
to the molecular mass of 25 kDa, which could be the probable chitinase protein which was
absent in the mutant.
Haggag (2002) worked with Trichoderma harzianum and T.koningii in view of
protecting tomato and cucumber plants from grey mould disease caused by Botrytis cinerea
through increasing the production of extracellular chitinase. Its synthesis was inducible by
mutagenising the isolates with 50 and 75 Kilo-rad doses of gamma irradiation which
resulted in the isolation of four mutants each of T. harzianum (TH12, TH18, TH32 and
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TH53) and T. koningii (TK5, TK15, TK24 and TK45) capable of producing high level of
chitinase. These mutants were stable and superior to the wild type (WT) with respect to
growth, sporulation and biocontrol potential against B. cinerea. From these mutants, two
mutants of each Trichoderma spp. (TH12, TH18, TK5 and TK15) produced high levels of
isozyme bands of chitinase and were aggressive than wild type and other mutants in
antagonizing B. cinerea.
Gohel et al. (2004) mutated Pantoea dispersa using physical and chemical
mutagens. Ultraviolet and gamma rays were used as mutagens separately for wild type
strain and EMS (chemical mutagen) was used for the further mutation of the mutant
obtained from the physical mutagenesis. Mutants were screened as chitinolytic producers
on the basis of zone of clearance on chitin agar plates incorporated with calcofluor white
M2R for better resolution. The mutants (no.8 & 10) were found to produce higher protease
and β1,3 Glucanase as compared to the wild type. The mutant strains were further used in
studies involving control of plant pathogens Fusarium sp. and Macrophomina phaseolina
(Tassi) and found to exhibit better inhibition percentage when compared to the wild strain.
2.6 Bioformulations and their Performances
Formulation plays a significant role in determining the final efficacy of a Bacillus-
based product, as do the processes of discovery, production, and stabilization of the biomass
of the biocontrol agent.
The export oriented agricultural and horticultural crops depends on the export of
residue free produce and has created a great potential and demand for the incorporation of
biopesticides in crop protection. To ensure the sustained availability of biocontrol agent’s,
mass production technique and formulation development protocols has to be standardized to
increase the shelf life of the formulation. It facilitates the industries to involve in
commercial production of plant growth promoting rhizobacteria (PGPR). The process of
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biopesticides development to complete product requires research in areas of screening,
formulation, field application, production, storage, toxicology as well as the steps necessary
for commercialization, such as scale up production, registration and regulatory matters
(Nakkeran et al, 2005). The major constraint for extensive use of biological control under
field conditions is a lack of knowledge concerning how to mass produce and properly
deliver biocontrol agents (Papavizas, 1985).
Mycolytic enzyme based formulations consisting of chitinolytic enzyme: protease
and glucanase have been used to control fungal plant pathogens (Deshpande, 1999).
Optimization of product and its formulation are most critical aspects to translate laboratory
scale activity into adequate field performance for any crop-protection agent. The
formulation must be user friendly which has to fulfill several criteria including allowing a
microorganism to retain and express its fungicidal properties; providing a significant
extension of shelf life. Moreover, the critical important step which is facing problem in
industrial scale is to harness the organism to industrial process of mass production
especially in fermentation as well as incorporation into user-friendly formulations (de-Vrije
et al., 2001).
These biocontrol agents can also be formulated by simple methods. Workers have
used various agents for formulation of Trichoderma such as peat, vermiculite, Koalin,
bentonite, lignite, molasses, cellulose granules, diatomaceous earth, wheat bran, charcoal
(Singh, 2003; Prasad and Rangeshwaran, 2000). Thus, culturing and maintaining biocontrol
agents is inexpensive and uncomplicated.
The work by Kavitha et al. (2005) with the isolate CBE4 and its formulation,
recorded a least per cent disease incidence of 15.66 followed by 16.07% in treatment with
BCA+. Significant increases in plant growth were induced by treatment with PGPR isolates
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CBE4 and BCA+. The efficacies of these PGPR isolates were found to be equivalent to the
standard fungicide Ridomil.
Mirik et al. (2008) isolated 3 Bacillus strains from soil samples of the rhizospheres
of peppers grown in greenhouses and fields to suppress the size of the population of X.
axonopodis pv. vesicatoria. Results indicated that disease development decreased by 11%-
62% and 38%-67% in pepper plants inoculated with the 3 Bacillus strains alone and in
combination, respectively, in greenhouse and field experiments. In addition, stem diameter,
root elongation, root dry weight, shoot dry weight, and yield increased in response to the
treatments in the field experiment by 7.0%-20.5%, 7.0%-17.0%, 4.5%-23.5%, 16.5%-
38.5%, and by 11.0%-33.0%, respectively.
Yanez-Mendizábal et al. (2012) reported that the Spray-dried formulations of
B.subtilis CPA-8 stored at 4 ± 1 and 20 ± 1°C showed good shelf life during 6 months, and
viability was maintained or slightly decreased by 0·2–0·3-log. CPA-8 formulations after 4-
and 6 months storage were effective in controlling brown rot caused by Monilinia spp. on
nectarines and peaches resulting in a 90–100% reduction in disease incidence.
2.7 Pot studies
The plant, pathogen and antagonists are co-exposed to controlled environmental
conditions. Exposure of the host to the heavy inoculum pressure of the pathogen along with
the antagonist will provide ecological data on the performance of the antagonist under
controlled conditions. Promising antagonists from controlled environment are tested for
their efficacy under field conditions along with the standard recommended fungicides. Since
the variation in the environment under field condition influence the performance of
biocontrol agent, trials on the field efficacy should be conducted for at least 15 – 20
locations under different environmental conditions to promote the best candidate for mass
multiplication and formulation development (Jeyarajan and Nakkeeran, 2000).
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Biocontrol preparation of both fungi and bacteria have been applied to seeds,
seedlings and planting media to reduce tomato wilt disease in the green house and field
condition with various degrees of success. The enzymes and antibiotics produced by
Trichoderma species and other biocontrol agents are strongly influenced by the substrate on
which the agent is grown and conditions in the laboratory may occur seldom in nature or
may not occur at all (Howell, 2003). Field trials are necessary to evaluate biocontrol agents
and thereafter repeated application of the agent on the selected field may be required to
obtain desired results. The success of biocontrol agents is greatly influenced by the
competence of pathogen to establish itself in soil and the opportunity to infect the host plant.
They are delivered either through seed treatment, bio-priming, seedling dip, soil application,
foliar spray, fruit spray, hive insert, sucker treatment and sett treatment. Kraft and Wilkins
(1989) studied severity of Fusarium disease in terms of soil compaction.
In the work of Suryanto et al. (2010), relative reduction of the seedling damping-off
was observed in all bacterial treatment ranged from 28.57 to 60.71%. Furthermore,
manifestation of bacterial suppression to Fusarium wilt was also exhibited by increasing of
seedling height (ranged from 7.33 to 7.87 cm compared to 6.88 cm) and dry-weight (ranged
from 2.7 to 4.3 mg compared to 2.3 mg).
Kamil et al. (2007) worked with chitinolytic bacteria and out of all the isolates
obtained, B. licheniformis (MS3) significantly reduced the damping off disease caused by
Rhizoctonia solani, in Helianthus annus using the seed coat or soil draining treatments
under green-house conditions.
Saman, (2007) screened Rhizobacteria in dual Petri plate assays to select
antagonistic strains against F. solani f. sp. phaseoli. The efficacy of the obtained isolates, B.
subtilis CA32 and the T. harzianum RU01 were tested in greenhouse pot experiments
against F. solani f. sp. phaseoli. Seed bacterization with B. subtilis CA32 and T. harzianum
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RU01 significantly protected bean seedlings from F. solani f. sp. phaseoli compared to the
untreated control plants. Plant protection was more pronounced in T. harzianum RU01
treated plants than bacterized plants.
Akhtar et al. (2010) examined the effects of Bacillus pumilus, Pseudomonas
alcaligenes, and Rhizobium sp. on wilt disease caused by Fusarium oxysporum f. sp. lentis
and on the growth of lentil. Inoculation with B. pumilus together with P. alcaligenes caused
a greater increase in plant growth, number of pods, nodulation, and root colonization by
rhizobacteria, and also reduced Fusarium wilting to a greater degree than did individual
inoculation. Use of Rhizobium sp. resulted in a greater increase in plant growth, number of
pods, and nodulation, and reduced wilting more than B.pumilus did. Combined application
of B. pumilus and P. alcaligenes with Rhizobium sp. resulted in the greatest increase in plant
growth, number of pods, nodulation, and root colonization by rhizobacteria, and also
reduced wilting in Fusarium-inoculated plants.
Yun Chen et al. (2012) evaluated the biocontrol ability of the 20 wild isolates
(CYBS-1 to CYBS-20) together with two model laboratory strains, the wild strain 3610 and
the domesticated strain PY79. The biocontrol efficacy of these strains was measured against
tomato wilt disease caused by a soil-borne plant pathogen R. solanacearum under
greenhouse conditions, The isolates showed a strong capacity to reduce the wilt disease
intensity, although the efficacy varied significantly among the strains (from 13.3% to 80%).
Six wild isolates (CYBS-5, -6, -12, -13, -14 and -19) as well as the model strain 3610 were
particularly effective in protecting against R. solanacearum, all achieving more than 50%
biocontrol efficacy (Xue et al., 2009).
Huang et al. (2012) used Micrographs to investigate the ability of Bacillus pumilus
(B. pumilus) SQR-N43 to control Rhizoctonia solani (R. solani) Q1 in cucumbers. The root
colonization ability of B. pumilus SQR-N43 was analyzed in vivo with a green fluorescent
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protein (GFP) tag. A pot experiment was performed to assess the in vivo disease-control
efficiency of B. pumilus SQR-N43 and its bio-organic fertilizer. Results indicate that B.
pumilus SQR-N43 induced hyphal deformation, enlargement of cytoplasmic vacuoles and
cytoplasmic leakage in R. solani Q1 mycelia. In the pot experiment, the biocontrol reduced
the concentration of R. solani. In contrast to applications of only B. pumilus SQR-N43 (N
treatment), which produced control efficiencies of 23%, control efficiencies of 68% were
obtained with applications of a fermented organic fertilizer inoculated with B. pumilus SQR-
N43 (BIO treatment).
The aim of the study by Chowdhury et al. (2013) was to evaluate the rhizosphere
competence of the commercially available inoculant Bacillus amyloliquefaciens FZB42 on
lettuce growth and health together with its impact on the indigenous rhizosphere bacterial
community in field and pot experiments. Results of both experiments demonstrated that
FZB42 is able to effectively colonize the rhizosphere (7.45 to 6.61 Log 10 CFU g−1 root
dry mass) within the growth period of lettuce in the field. The disease severity (DS) of
bottom rot on lettuce was significantly reduced from severe symptoms with DS category 5
to slight symptom expression with DS category 3 on average through treatment of young
plants with FZB42 before and after planting.