REVIEW OF LITERATURE - Information and Library …shodhganga.inflibnet.ac.in/bitstream/10603/45223/7/07_chapter 2...seedling damping-off, late blight, downy mildews, and white rust

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



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);



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



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



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



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.



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.



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.



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



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



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).



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



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



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



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



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-



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.



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



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



(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.



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.



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%



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



(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),



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).



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



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-



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.



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



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.



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



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-



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



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



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



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).



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



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



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.

Documents

REVIEW OF LITERATURE - Information and Library …shodhganga.inflibnet.ac.in/bitstream/10603/45223/7/07_chapter 2...seedling damping-off, late blight, downy mildews, and white rust