5

CHAPTER 2

LITERATURE REVIEWS

2.1 Plant Growth Promoting Rhizobacteria

The term of plant growth promoting rhizobacteria (PGPR) was used originally to

describe this unique biological control groups of microorganisms. Generally, PGPR

can be classified into two groups; biocontrol plant growth promoting rhizobacteria

(PGPR) and the plant growth promoting rhizobacteria (PGPR) (Basen and de Bashan,

2002).

2.1.1 Biocontrol PGPR

Biocontrol PGPR are the microorganisms that suppress plant pathogens by

producing variety of inhibitory substances or by increasing of the natural resistance of

plants or by displacing the pathogen (Bashan and de Bashan, 2002).These

microorganisms rapidly colonize the rhizosphere and combat the soil borne pathogens

at the surface of root (Rangarajan et al., 2003). Some of these methods of the

biocontrol PGPR are discussed below.

2.1.1.1 Antibiosis

The biocontrol PGPR are endowed with the capacity to produce antibiotics

against a number of phytopathogenic fungi such as 2,4-diacetylphloroglucosinol

(2,4-DAPG), phenazine, pyrrolnutrine, and pyluteolin (Rangarajan et al., 2003).

Hydrogen cyanide (HCN) is also antifungal metabolites produced by these microbes.

More recently the production of new antifungal metabolites belonged to the class of

6

cyclic lipopeptides; visconsinamide and tensin have been reported (Bloemberg and

Lugtengerg, 2001).

Pseudomonas sp. and Bacillus sp. have been widely investigated; several

strains of Pseudomonas are used to control diseases in a variety of crop and other

non-crop plants (Commare et al., 2002). During their stationary growth phase, the

biocontrol strains of Pseudomonas synthesize the antibiotics phenazine carboxylic

acid (PCA), 2,4-DAPG, pyoluterin, and pyrrolnitrin (Bora et al.,2004). Many of these

antibiotics produced by Pseudomonas spp. in-situ contributed to the suppression of

many plant diseases. Plant diseases caused by Pythium ultimum and Rhizoctonia

solani are suppressed by different strains of Pseudomonas fluorescens (Cheryl et al.,

1998). Pytium ultimum, the causative agent of causing damping off in sugar beet,

inhibits the production of 2,4-diacetylphloroglucinol(2,4-DAPG) by P. fluorescens

F111 (Dikin et al., 2007). This product is produced by other P. fluorescens strains

and has been found effectively against Fusarium oxysporum attacking tomatoes.

Recently, it has been demonstrated that fluorescent Pseudomonas producing the

2,4-DAPG plays as a key role in the suppresiveness of take-all decline (TAD) in soils

(de Souza et al., 2003a).

Phenazine is a secondary metabolite effectively against phytopathogenic

fungi. Pseudomonas which produce phenazine is reported for its suppression of take

all of wheat caused by Gueumanomyces graminis var tritici (Chin-A-Woeng et al.,

2002).

7

Cyclic lipopeptides such as viscosinamide produced by P. fluorescence have

been shown to control Pythium and Rhizoctonia solani. Cyclic lipopeptides induced

encystment of Pythium zoospores and affected the mycelia of R. solani and

P. ultimum by causing reduced growth and intracellular activity, hyphal swelling, and

increased branching (de Souza et al., 2003b).

The next most widely researching and commercial bacteria for biocontrol

activity in soil are member of the genus Bacillus. Most of the antibiotics produced by

Bacillus were found to be peptides (Commare et al., 2002). Bacillus cereus UW 85

produces the antibiotic zwittermycin A and antibiotic B which tend to suppress

damping off disease and root rot of soy beans (Emmert and Handelsman, 1999).

The other members of this genus, Bacillus subtilis which is the most importance

species. The most commercially successful strain is B. subtilis GBO 3. This strain

effectively colonized plant roots and produced antifungal compounds and is an active

ingredient in one of the widely distributed biofungicides (McSpadden and Fravel,

2002). Another useful biocontrol strain in Australia is B. subtilis A13 (Kim et al.,

1997). This strain was shown to inhibits all of the nine pathogens tested in in vitro,

subsequently promotes the growth of cereals, sweet corn, and carrot when applied as

seed inoculants, therefore, Bacillus spp. are considered ideal candidates used for

biocontrol agent in seed treatment against soil borne pathogens.

8

2.1.1.2 Induction of Systemic Resistance

Induced Systemic Resistance (ISR) is the system that protects a plant using

inducing agents that stimulate resistance by activates genes that encode chitinase,

peroxidase, ß-glucanase and enzymes involved in the synthesis of phytoalexins

(Ona et al., 2005). ISR is a new topic in disease suppression. Biocontrol PGPR elicit

ISR in plants through fortifying the physical and mechanical strength of the cell wall

as changing the physiological and biochemical reaction of host plant. This leads to the

synthesis of defence chemical against the challenging pathogen (Rammamoorthy

et al., 2001). The other mechanism by ISR is the production of PR-protein

(Pathogenesis related proteins) such as chitinase, peroxidase, synthesis of phytoalexin

and other metabolites (Ona et al., 2005). Increased expression of plant peroxidase and

chitinase in rice, stimulated by P. fluorescence was efficient to inhibit mycelial

growth of the sheath blight fungus R. solani (Nardakumar et al., 2001). Seed

treatment of pea by P. fluorescence results in the production of ß-glucanase and

chitinase (Benhamou et al., 1996b). In all cases of the host lytic enzymes accumulate

at the site of penetration of the fungus. Pseudomonas mediated ISR against

F. oxysporum was also observed in carnations (van Per et al., 1999), and against

Collectotrichum orbicalare and Pythium aphanidermatum infection of cucumber

(Chen et al., 2000). Another important aspect of ISR is the synthetic protection of

plants following application of an inducing agent.

9

2.1.1.3 Competition in The Rhizosphere

The ability to compete nutrients by indigenous microbial populations is an

important trait for biocontrol of soilborne pathogens. Pseudomonas spp. have been

reported to have the ability to metabolize the constituents of seed exudates in order to

produce compounds inhibitory of Pythium ultimus (Bora et al., 2004). Due to

competition, biocontrol agents have the ability to displace some bacterial plant

pathogens. Cooksey (1999) used co-inoculated a non-pathogenic copper resistant

mutant of P. syringae, the casual agent of bacterial speck of tomato, with a pathogenic

strain with the causal a surfactant decrease the disease incidence by competing with

the pathogen for the same niche.

2.1.1.4 Siderophore Production

Biocontrol PGPB prevents the soilborne pathogens by production and

secretion of siderophores. These molecules bind most of Fe+3available in the

rhizosphere thereby effectively preventing any fungal pathogens due to a lack of iron.

Biocontrol PGPB out of competition with fungal pathogens due to a lack of iron

(Chincholkar et al., 2007a) and out of complete fungal pathogens for the available

ferric iron in the rhizosphere. Siderophores also indirectly stimulates the biosynthesis

of the other antimicrobial compounds by making these minerals easily availale to

bacteria (Dikin et al., 2007).

The major types of siderophore produced by PGPR include pyoverdin,

pyochelin and salicylic acid (Bultreys and Gheysen, 2000).The fluorescent

Pseudomonas species are an efficient competitor for ferric iron (Fe+3).The most

commonly detected siderophore in these species are pyoverdin or pseudobactins

(Lemanceau et al., 1993). Many potential biocontrol strains of this species produce

10

pyoverdin. They are generally be peptide siderophores and all containing the same

quinoline chromophore which are responsible for the color of molecule, a peptide

chain and a dicarboxylic acid connected to the chromophore. The characteristic

fluorescent pigments of Pseudomonas are due to the pyoverdin (Bultrey et al., 2003).

This compound make ferric iron unavailable to pathogen in the rhizosphere

(Bora et al., 2004).

2.1.2 Plant Growth Promoting PGPR

Plant growth promoting PGPR are microorganisms which promote growth via

production of phytohormones and the improvement of plant nutrition status.

Alternatively, PGPR may increase plant growth in the other ways, for example, by

associatied with N2 fixation, solubilizing nutrients such as P, promoting mycorrhizal

function, regulating ethylene production in roots, releasing phytohormones and

decreasing heavy metal toxicity. Some of these modes of action are discussed below.

2.1.2.1 Solubilization of Inorganic Phosphates

In recent years, great attention has been dedicated to the study of the role of

soil microorganisms play in the dynamics of phosphate (P), particularly then ability to

solubilize insoluble P forms. These microorganisms are bacteria and fungi that inhabit

the rhizosphere (Bowen and Rovira, 1999). Most soil bacteria can solubilize insoluble

phosphates; particularly active are those that belong to the genera Pseudomonas,

Enterobacter, and Bacillus as well as some soil fungi such as Penicillium and

Aspergillus. The mechanisms involved in the microbial solubilization of P are the

production of organic acids and the release of protons to the soil solution.

11

Inoculation with PSRB and other soil microorganisms, such as arbuscular mycorrhizal

fungi (AMF), might enhance even more the benefits of this P solubilization

(Whitelaw, 2000).

Phosphorus (P) is one of the major essential macronutrients for biological

growth and development. Phosphorus plays an indispensable biochemical role in

photosynthesis,respiration,energy storage and transfer, cell division, cell enlargement,

and several other processes in the living plant. It is presented at levels of 400-1200

mg kg-1of soil. Microorganisms play a central role in the natural phosphorus cycle.

This cycle occurs by the cyclic oxidation and reduction of phosphorus compounds,

where electron transfer reactions between oxidation stages range from the phosphine

(-3) to phosphate (+5). The concentration of soluble P in soil is very low, normally at

the level of 1 ppm or less than 10 M H2PO4- (Lal, 2002).

The biggest reserves of phosphorus are rocks and other deposits, including

primary apatites and other primary minerals formed during the geological age.

Mineral forms of phosphorus are represented in soil by primary minerals, such as

apatite, hydroxyapatite, and oxyapatite. They are found as a part of the stratum rock,

and their principle characteristic is their insolubility. Mineral phosphate can be also

found associated with the surface of hydrated oxides of Fe, Al, and Mn, which are

poorly soluble and assimilable. This is characteristic of ferralitic soil, in which

hydration and accumulation of hydrated oxides and hydroxides of Fe take place,

producing an increase of phosphorus fixation capacity (Lal, 2002).

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Most agricultural soils contain large reserves of phosphorus, a considerable

part of which has accumulated as a consequence of regular application of P fertilizers

(Chung et al., 2005). However, a large portion of soluble inorganic phosphate is

applied to soil as chemical fertilizer but is rapidly immobilized after application and

becomes unavailable to plant. The fixation and precipitation of P in soil are dependent

on pH and soil type. In acid soil, phosphorus was fixed by free oxides and hydroxides

of aluminum and iron, while in alkali soil it was fixed by calcium, causing a low

efficiency of soluble P (Lal, 2002).

Because of the problem of ‘P’ availability to plants, there is now a growing

need in the selection of biofertilizer in plant nutrition. The mechanism by which these

microorganisms to solubilize insoluble P is decreases the pH of surroundings either by

the release of organic and or protons (Gyanshewar et al., 2002).

Species of Pseudomonas and Bacillus are reported as the most important

phosphate solubilizers. For example, P. putida, stimulated the growth of roots and

shoots and increased P uptake in canola. Inoculation of crop with B. firmus and

B. polymyxa also resulted in phosphate uptake and yield increase (Khan et al.,2006).

Among these, B. megaterium is regarded as the most effective Phosphate Solubilizing

Microorganism (PSM) in many field experiments to release ‘P’ from organic

phosphate, but does not solubilize mineral phosphate (Gyanshewar, 2002). Phosphate

solubilizing bacteria are also reported to function as mycorrhizal helper bacteria.

When such bacteria are associated with mycorrhizal fungi, they promote root

colonization. The principle was that, their association with mycorrhizal fungi

contributes to the biogeochemical cycle of nutrients by more than providing a greater

surface area for scavenging nutrients that may be relatively immobilize in soil

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(Niranjan, 2006). Generally, the role of microorganisms, especially of the growth

promoting rhizobacteria in ‘P’ solubilization and mineralization is very crucial to

make ‘P’ easily available to plants.

2.1.2.2 Synthesis of Phytohormones

A great proportion of microorganisms capable of producing in vitro

phytohormones is found to survive in the rhizospheric. According to this finding, 20%

of the bacteria produced phytohormones. The auxin type phytohormone known as

indole-3-acetic acid (IAA) is the main type produced by plant growth promoting

bacteria (PGPB) (Patten and Glick, 2002).Beneficial bacteria synthesize IAA through

the indole-pyruvic pathway. In this pathway, amino acid tryptophan is the first

transformed into indole-3-pyruvic acid by oxidative deamination, which is then

decarboxylated into indole-3-acetaldehyde. Indole-3- acetaldehyde is finally oxidized

to IAA (Patten and Glick, 2002).

Among the most efficient PGPB studied for their capacity to produce

phytohormones are P. putida, P. fluorecens, Azospirillum spp. and Bacillus spp.

In all these bacteria, Pseudomonas the formation of IAA and other auxins have been

proved using HPLC and mass spectrometry (Idris et al., 2007).

Recently, the role of many rhizobacterial produces IAA in the development

of the host plant root system has been studied, for example, canola seeds treated with

a wild type of P. putida strain that produces IAA and other IAA deficient mutant

constructed by insertional mutagenesis response differently (Patten and Glick, 2002).

The primary roots of canola seeds treated with the wild type strain were on the

14

average longer than the roots from seeds treated with a mutant strain and root from

un-inoculated seeds. It was suggested by the other studies that, low levels of IAA

stimulated primary root elongation but high levels of IAA stimulated the formation of

lateral and adventitious roots (Ona et al.,2005).

2.1.2.3 Asymbiotic Nitrogen Fixation

To order on sustainable of sufficient crop production, a reliable source of

nitrogen is vital. Microbial oxidation of soil organic matter may provide plants with

potentially available nitrogen. However in soils with low soil organic nitrogen pool, in

the rhizospheric soil, free living nitrogen-fixing PGPR affect plant growth directly by

non-symbiotic nitrogen fixation (Bora et al., 2004).

Many non-legume plants have been shown to be associated with the free

living diazotrophic bacteria. With the advent and the application of acetylene

reduction assay, it has become a common practice to screen plants and

microorganisms for the presence of nitrogenase activity. In ecosystem where legumes

are absent, nitrogen fixation by free living diazotrophic bacteria is the mechanism to

meets part of the nitrogen requirement of the plants (Jousset et al., 2006). Free living

nitrogen fixers in the rhizospheric soil are nowadays given attention as they are

known for the utilization of plant exudates as a source of energy to support the

nitrogen fixing process.

Azospirillum proliferate in the rhizospheric soil of many tropical grasses,

fixing nitrogen and transferring it to the plants. Field inoculation with Azospirillum in

many investigations revealed that this bacterium is capable of promoting the yield of

15

many important agricultural crops such as rice (Malik et al., 1997), corn (Zea mays),

sorghum (Sorghum bicolor) and switch grass (Pinatum virgutum) (Bredjda et al.,

1994).

The auxin type phytohormones produced by Azospirillum spp. affect root

morphology and improve nutrient uptake from soil (Barea et al., 2005). Apart from

increasing the density and length of legume root hairs, IAA secreted by Azospirillum

increases the amount of flavonoids that are secreted and act as signals for initiations

of root nodulation by rhizobial strains (Glick et al., 2001).

2.2 Antifungal Antibiotics

Natural products are an important source of new bioactive compounds and for

the drugs launched over the period of 1981-2002. In agriculture, Dikin et al., (2007)

estimated that natural products had only about 10% of the market for crop-protection,

and thus, the share for antifungal antibiotics was even smaller. Research on new

agents for controlling phytopathogenic fungi has been a concern for the agriculture

industry. The factors for screening of antifungal compound are described as follows.

2.2.1 Microbial Sources

Organisms that inhabit unique niche or area of high biodiversity should have a

high probability of producing unique metabolites. Some of these like species of

Streptomycetes and Pseudomonas, are capable of producing a spectrum of chemically

diverse metabolites, and can be said to be metabolically talented. By changing the

16

culture conditions for such strains, different metabolic outputs can be obtained, and

even new compounds can be isolated.

2.2.2 Structural Diversity of Antifungal Antibiotics

Antifungal antibiotics display a wide structural diversity and their modes of

action target a number of fungal enzymes and physical structures. In the overview,

they are presented according to a compound class.

Carbohydrates

Figure 2.1 Kasugamycin

The disaccharide kasugamycin (Figure 2.1) was isolated from Streptomyces

kasugaensis in 1965; it blocks protein synthesis and shows low toxicity to humans.

Kasugamycin is used for treatment of Pyricularia oryzae but as the pathogen rapidly

develops resistance to the compound, it is used combination with other fungicides

(Copping, 2007).

Amino Acids and Peptides

A large group of cyclic peptides, lipopeptides, and lipodepsipeptides have

been isolated on their antifungal activity, and they act by several different modes of

action. The bacterium Pseudomonas syringae pv. syringae has been the source of a

number of lipodepsinonapeptides: syringomycins, syringotoxins,

syringostatins,

and

17

pseudomycins. They are thought to interact with the fungal cell membrane by pore

formation, which results in fatal electrolytic leakage (Jousset et al., 2006).

Terpenes

A group of polar triterpenes were reported in the 2000. They include the

fungal metabolites enfumafungin from a Hormonema sp.,

ascosteroside from

Ascotricha amphitricha, arundifungin from Arthrinium arundinis and ergokonins from

Trichoderma koningii (Reino et al., 2007).

Polyketide

Polyketide macrolides and polyene macrolides have been extensively

investigated for their antifungal activity and the latter contain some of the most

important antifungal drugs for human use e.g. amphotericin B and nystatin.

Fatty Acids and Fatty Acid Mimics

Several structures with fatty acid or fatty acid-like moieties have been isolated

as antifungal, specifically in conjunction with screening for inhibition of enzymes

connected with sphingolipid biosynthesis. These include viridiofungins from

Trichoderma viridae (Reino et al., 2007).

Nucleoside Analogues

For example, Polyoxin A was isolated in 1965 on a sample from Streptomyces

cacaoi strain. It selectively inhibits the fungal enzyme chitin synthetase, which

polymerizes N-acetylglucosamine to chitin. This specific mechanism of the polyoxins

18

is highly interesting for drug development due to chitin is not present in human cells.

In agriculture, the polyoxins are effective for treatment of a variety of fungal

pathogens, especially Alternaria spp., Botrytis cinerea and Pyricularia oryzae

(Lamberth, 2010).

Other Biosynthetic Origin

The small tryptophan derived antifungal antibiotic pyrrolnitrin (Figure 2.2)

was isolated in 1964 from a strain of Pseudomonas pyrrocina. Pyrrolnitrin acts by

inhibiting mitochondrial respiration (Seibold et al., 2006).

Figure 2.2 Pyrrolnitrin

The strobilurins inhibit mitochondrial respiration in fungi and were found to

control several important plant pathogens. The strobilurins are at present an important

group of agricultural fungicides (Herms et al., 2002).

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Figure 2.3 Antifungal antibiotics grouped according to fungal target (Siddiqui, 2006)

2.3 Isolation of Antifungal Antibiotics

2.3.1 Bioassays

Bioassays are characterized as in vivo and in vitro. The in vitro assays can be

separated into diffusion and dilution assays (Jousset et al., 2006). In diffusion assays,

the sample needs to diffuse through a solid medium which also contains the test

organism. The classical diffusion assay consists of placing a paper disc soaked with

the test compound on an agar plate inoculated or plated with fungal spores, or

20

pipetting a solution of the sample to a small well in the agar plate. The size of any

mycelium-free zone around the sample after inoculation is used as the bioassay

reading. Agar is compatible with hydrophilic and moderately lipophilic compounds

but highly lipophilic compounds and charged compounds do not diffuse well.

For lipophilic compounds normal phase TLC plates have been used as solid support

(Arikan et al., 2001),

although it has been proposed that minimal inhibitory

concentration (MIC) values from this method might be overestimated in comparison

with other assays. Diffusion assays are very versatile and do not require expensive

equipment. However, the observed biological activity is hard to correlate to exact

concentrations of the test compounds as the inhibition zones depend on concentration

gradients. The relative large size of the individual assays also makes it laborious to

test many samples. In dilution assays the test organism is suspended in a solution

where the sample concentration can be controlled. Solubility of the samples may pose

a problem and can be improved by additions of detergents or organic solvents at

levels that do not affect the assay organism too much. Dilution assays are easily

adapted to automation and can be used with micro liter plates in the 96, 384, and even

1536 format. Most assays belong to this category, as well as the dilution micro liter

bioassay used in the work of this thesis.

3.2 Sample Preparation and Isolation

Bioactive natural products are often presented in low concentrations.

In extreme cases, metabolites can accumulate up to a few percent of the organisms

dry weight or growth medium, but the concentrations are often much lower, usually at

the ppm or sub-ppm level. Different types of sample preparation are needed for

removing the bulk matrix and concentrating the metabolites prior to chromatographic

21

separation.

Traditionally, liquid-liquid extraction has been used for extracting

metabolites and the method is well suited for working with extracts from a solid or

liquid matrix. The main drawbacks are high consumption of organic solvents and

difficulties in automation. When working with liquid microbial cultures, solid phase

extraction (SPE) is a good alternative.

Solid Phase Extraction

Solid phase extraction (SPE)

relies on the same principles as all other

chromatographic techniques; the distribution of the analysis between a stationary

phase and a mobile phase. The solid phase comes in pre-packed disposable columns

and in bulk for packing columns of desired size. The aqueous sample solution is

passed through the column and the analytes are adsorbed on the stationary phase.

The material is then desorbed with a small amount of organic solvent. This results in a

concentrated lipophilic fraction and a hydrophilic fraction with the same size as the

loading volume. SPE has very low resolution when compared to HPLC and is used as

a kind of digital or discrete chromatography. The column is then eluted with different

solvents, and the adsorbed compound is either displaced or is still bound to the

column. Any intermediate results are unwanted. The technique is easily automated

and uses of small amounts of organic solvents (Rossi and Zhang, 2000).

22

High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is a standard analytical and

preparative technique for fractionating complex samples to obtain pure analysts.

Separation of samples in HPLC depends on the choice of stationary phase, mobile

phase, temperature,and flow rate. The C18

stationary phase is a default starting point in

the sample isolation. The mobile phase is usually a combination of water and a

miscible organic solvent such as MeOH or MeCN. A common modification of the

mobile phase is control of pH by acids or bases and organic or inorganic buffers.

The HPLC system can be coupled to a number of detectors depending on the type of

samples analyzed. Common detectors are refraction index (RI), UV/VIS, and MS

detectors, of which the two latter are often combined (Tang et al., 2005).

Purification of complex samples on HPLC is started with a standardized

gradient running that will give a coarse fractionation of the sample based on

lipophilicity. The next HPLC step is concentrated on developing an isocratic or

gradient method that will separate the compounds into pure fractions.

2.3.3 Structure elucidation

In natural product chemistry, NMR and MS are often sufficient to determine

the compound skeleton. IR, which can identify functional groups and carbon

hybridization level, has been phased out, and is mainly used as a compound

characterization method, together with optical rotation, UV absorbance, and melting

23

point. In the determination of absolute configuration, the method of choice is

dependent on the properties of the investigated compound. A short presentation of

MS, NMR, and two approaches to determine absolute configuration is listed below.

Mass Spectrometry

In mass spectrometry (MS) the investigated species is ionized and detected by

its specific mass to charge ratio (m/z).The output data give information on the

monoisotopic mass of the investigated species. If the ionisation technique is soft,

likes in electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation

(MALDI),the molecular ion is present at high intensity.Soft ionisation is often

combined with one or several fragmentation step on selected molecular ions to yield

more information on the species. In high energy ionisation likes electron impact (EI),

most molecular ions are fragmented, and the fragmented ions are presented in the

mass spectrum. The high fragmentation pattern reproducibility in EIMS makes it

possible to use the compound mass spectrum as a fingerprint.

If the mass analyzers have sufficiently high resolution, the data can be used to

calculate the elemental composition of the species, because of the minute differences

in mass of neutrons and protons, together with the small mass addition of electrons.

Mass analyzers with high resolution include sector instruments, some time-of-flight

(TOF)-instruments, and the gold standard Fourier Transform Ion Cyclotron

Resonance (FT-ICR) instruments. The mass spectrometer is usually associated to a

chromatographic system for separation of complex samples before MS analysis.

24

For HPLC, the standard is complied to an ESIMS while GC is compatible with EIMS.

Mass spectrometry is a destructive technique but most variants are very sensitive,

requiring only micrograms or nanograms for analysis.

In compound isolation and identification, mass spectrometry fills many

functions. It can be used for scanning the complex samples for specific compounds,

either directly in mixture, or by using the mass spectrometer as a detector in a

chromatographic system (HPLC-ESIMS, GC-MS). It can also indicate (ESIMS,

EIMS,MALDITOF) or verify (high resolution techniques) compound elemental

composition.

Nuclear Magnetic Resonance Spectroscopy

Certain nuclei have a magnetic spin, which can be manipulated by an external

magnetic field. This is suitable for using of nuclear magnetic resonance (NMR)

spectroscopy. The spin possessing nuclei of main interest in natural product structure

elucidation are 1H,

13C,

15N and to some extent

31P. By using one and two-dimensional

NMR techniques the carbon-hydrogen bonding pattern can be elucidated. NMR is a

non-destructive technique but sensitivity is lower than MS. One way to achieve higher

sensitivity is to use a stronger magnetic field. At present, field strengths

corresponding to a proton frequency of 400-900 MHz are widely used. If the sample

tube is narrow, the sensitivity will be high, as less solvent can be used and the

receiving coils can be placed closer to the sample. Use of a cryo-probe, which reduces

noise by cooling the electronics, will also boost sensitivity significantly. With a 600

25

MHz NMR spectrometer, equipped with a 2.5 mm non-cryo microprobe (45 μl

sample volume), less than a micromole is sufficient for elucidation.

A standard set of NMR experiments is normally used to elucidate an unknown

structure. One-dimensional1H NMR gives a picture of the hydrogens of the sample

and proton-proton coupling constants. Two-dimensional homonuclear (1H,

1H)

experiments show proton spin systems (COSY and TOCSY experiments) or can

identify through-space correlations between protons (ROESY and NOESY

experiments).Two-dimensional heteronuclear (1H,

13C) NMR will display one-bond

correlations between carbons and protons (HSQC experiments) or two-bond to four-

bond correlations between carbons and protons (HMBC experiments) (Reynolds and

Enriquez, 2002).

2.3.4 Factors Affecting Antibiotic Production, Activity, and Detection

The efficiency of recovery of antibiotics from natural sources is influenced by

their stability, chemical and physical interactions with the sample matrix and

the extraction solvent, and the handling of the sample before and during extraction.

The choice of extractant will depend on the solubility and charge properties of the

antibiotic, which can be predetermined empirically by processing cultures grown

in vitro. A procedure suitable for the extraction of many of the antibiotics produced

by fluorescent Pseudomonas spp. has been published (Dikin et al., 2007), and can

be adapted for other substances by adjusting the amount of sample required and

selecting appropriate solvents. This method can recover phenazine-1-carboxylic acid,

its hydroxyphenazine derivatives, pyrrolnitrin, pyoluteorin, and 2,4-DAPG, but not

26

for the phenazine compound pyocyanin, which has different solubility properties.

In general, samples (either hydrated or dry) are dispersed in the extractant; the solid

residues are removed by centrifugation, and the filtrate is collected and concentrated.

A wide range of ratios of sample mass to solvent volume has been reported,

but values of 1:1 to 1:5 are typical. Samples are fractionated by HPLC. Antibiotics

with ionizable residues can be separated from some contaminants by exploiting the

pH-dependent, differential solubility of the neutral and charged forms in organic and

aqueous solvents (Arikan et al., 2001). Most isolation procedures therefore include

at least one liquid-liquid extraction step to partition antibiotics away from salt

residues and impurities, and into organic solvents from which they can be

concentrated readily. Solid-phase extraction to recover bioactive compounds from

natural sources offers many advantages over liquid-liquid solvent partitioning. Less

solvent waste is generated, isolation is rapid and efficient, and sensitivity of detection

may be improved because trace substances in large volumes of solvent can be

enriched.

Aminoindoles and carboxylic indoles were recovered with high efficiency on

Amberlite XAD-2 and C18 columns, respectively (Belley et al., 2005), and

macrocyclic xanthobaccin compounds produced by Stenotrophomonas sp. strain

SB-K-88 in the rhizosphere of sugar beet were trapped by growing the seedlings in a

1:1mixture of sand and Amberlite XAD-2 resin (Nakayama et al., 2001).

Chromatography and detection Thin layer chromatography (TLC) are widely used to

fractionate antibiotics recovered from natural materials. Both normal and reversed-

phase adsorbents have been used with a variety of mobile-phase solvent systems.

Substances are visualized by UV absorption, chromogenic reaction with spray

27

reagents, or bioautography, in which suspensions of indicator organisms in agar or

broth are overlaid on chromatograms to detect bioactive spots (Tang et al., 2005).

Antibiotic identity is confirmed by appearance, distance travelled relative to the

solvent front (Rf value), and chromatography with standards in at least two different

solvent systems. Quantities are estimated from spot size and intensity, or size of the

inhibition zone for bioautography, at various dilutions relative to known amounts of

standards run on the same plate (Chin-a-Woeng et al., 2002).

The versatility, resolving capability, and quantitative accuracy offered by

HPLC make it be a method of choice for most analyses of antibiotics produced in situ.

HPLC is readily coupled with techniques such as mass spectrometry or NMR to

further resolve mixtures of related compound and to provide insight into chemical

structure (Chin-a-Woeng et al., 2002).Considerations in optimizing or developing a

chromatographic system include selection of the column, the mobile phase,

the elution profile, and the detector to be used. Reversed-phase columns have been

used almost exclusively for antibiotics produced in situ, and a variety of liquid phase

systems and elution profiles have been described. Detection most frequently is by

UV absorbance. Individual components within a mixture can be monitored

simultaneously, each at its own absorption maximum, and subsequent spectral

analyses can provide insight into peak purity and identity (Arikan et al., 2001).

Greater sensitivity and selectivity of detection can be obtained for some phenolic

compounds by amperometric and fluorometric detections may offer similar

advantages for compounds such as indoles (Belley et al., 2005) and some phenazines.

28

2.4 Delivery Systems

Plant growth promoting rhizobacteria (PGPR) are delivered through several

means based on the survival in nature and the mode of infection of the pathogen. They

are delivered through seed, soil, foliage, rhizomes, or through combination of several

methods of delivery as described below.

Seed Treatment

Seed treatment with cell suspensions of PGPR was effective against several

diseases. Delivery of Serratia marcescens strain 90-166 by seed dip before planting,

and the inoculated with soil of 100 ml of the same strain at the rate of 108 CFU ml-1

seeding reduced bacterial wilt of cucumber and controlled cucumber beetles,

moreover, increasing the fruit weight (Zhender et al., 2001).Transferring of

technology for commercial use could be possible,if PGPR strains are made available

as a product. After realization of the same, several carriers were used for formulation

development. Talc based formulation of P. fluorescens Pf1 was coated on chickpea

seeds at the rate of 4 g Kg-1(107 CFU g-1) for the management of chickpea

wilt.Sowing of treated chickpea seeds resulted in establishment of rhizobacteria in the

chickpea rhizosphere (Vidhyasekaran and Muthamilan, 1999). Treatment of cucumber

seeds with strain mixtures comprising of Bacillus pumilus-INR7,B. subtilis-GB03,

and Curtobacterium flaccumfaciens-ME1 at the a mean bacterial density of 5x109

CFU/seed reduced intensity of angular leaf spot and anthracnose equivalent to the

29

synthetic elicitor Actigard® and better than seed treatment with individual strains

(Gowen and El-Hassan, 2006).

Bio-priming

Bio-priming of seeds with bacterial antagonists increase the population load of

antagonist to a tune of 10-fold on the seeds thus protected the rhizosphere from the

ingress of plant pathogens(Callan et al., 1990). Drum priming is a PGPR formulations

265 commercial seed treatment method followed to treat seeds with pesticides. Drum

priming of carrot and parsnip seeds with P. fluorescens Pf CHAO proliferated well on

the seeds and could be explored for realistic scale up of PGPR (Whipps and Bennett,

2008).

Seedling Dip

PGPR are delivered by various means for the management of crop diseases

based on the survival nature of pathogen.In several crops pathogens gain entry into

plants either through seed, root or foliage. In rice, sheath blight incited by Rhizoctonia

solani is a major obstacle in rice production. Delivery of P. fluorescens strain

mixtures by dipping the rice seedlings in bundles in water containing talc based

formulation of strain mixtures (20 g l-1) for 2 h and later transplanting it to the main

field suppressed sheath blight incidence (Nandakumar et al., 2001).Dipping of

strawberry roots for 15 minutes in bacterial suspension of P. putida (2 x 109CFU ml-1)

isolated from strawberry rhizosphere reduced Verticillium wilt of strawberry by 11%

30

compared to untreated control (Berg et al., 2001). Dipping of Phyllanthus amarus

seedlings in talc based formulation of B.subtilis (BSCBE4) or P. chlororaphis (PA23)

for 30 minutes prior to transplanting reduced stem blight of P. amarus

(Mathiyazhagan et al., 2004).

Soil Application

Soil, being as the repertoire of both beneficial and pathogenic microbes,

delivering of PGPR strains to soil will increase the population dynamics of augmented

bacterial antagonists and thereby will suppress the establishment of pathogenic

microbes. Vidhyasekaran and Muthamilan (1997) stated that soil application of a

peat based formulation of P. fluorescens (Pf1), at the rate of 2.5 kg of formulation

mixed with 25 Kg of decomposed farm yard manure in combination with seed

treatment, increased the rhizosphere colonization of strain Pf1 and suppressed

chickpea wilt caused by Fusarium oxysporum f. sp. ciceris. Broadcasting of talc based

formulation of strain mixtures ( Pf 1 and FP 7 ) by blending 2.5 kg of formulation

with 50 kg of sand after 30 days of transplanting paddy seedlings to main field

significantly reduced sheath blight and increased yield under field conditions

(Nandakumar et al., 2001). Incorporation of commercial chitosan based formulations

LS254 (comprising of Paenobacillus macerans + B. pumilus) and LS255 (comprising

of P. macerans + B. subtilis) into soil at the ratio of 1:40 (formulation : soil) increased

bio-matter production by increasing root and shoot lengths and yield (Vasudevan et

al.,2002). Soil application of the strain mixture formulations LS 256 and LS 257

comprising of two different Bacillus spp., was better than seed treatment and

31

suppressed downy mildew under greenhouse and field conditions (Niranjan Raj et al.,

2003).

Foliar Spray

The efficacy of biocontrol agents for diseases is greatly affected by

fluctuations microclimate. The phyllosphere is subject to diurnal and nocturnal, cyclic

and non-cyclic variation in temperature, relative humidity, dew, rain, wind and

radiation. Application of P. fluorescens to foliage (1 kg of talc based formulation ha-1)

on 30, 45, 60, 75 and 90 days after sowing seed reduced leaf spot and rust of

groundnut under the field conditions(Meena et al.,2002).Pre-harvest foliar application

of talc based fluorescent Pseudomonas strain FP 7 supplemented with chitin at

fortnightly intervals (5 g l-1;spray volume 20 l-1tree) on to mango trees from pre-

flowering to fruit maturity stage of mango trees induced flowering to the maximum

and reduced the latent infection by C. gloeosporioides, and also increasing the fruit

yield and quality (Vivekananthan et al., 2004).

Sucker Treatment

Plant growth promoting rhizobacteria also play a vital role in the management

of soilborne diseases of vegetatively propagated crops. The delivery of PGPR varies

depending on the crop. In the crops like sugarcane and banana rhizobacteria were

delivered through set treatment or rhizome treatment respectively. Banana suckers

were dipped in talc based P. fluorescens suspension (500 g of the product in 50 l of

32

water) for 10 min after pairing and pralinage. Subsequently, it was followed by

capsule application (50 mg of P. fluorescens per capsule) on third and fifth month

after planting. It resulted in 80.6 percent reduction in panama wilt of banana

compared to the control (Ramamoorthy et al., 2001).

Multiple Delivery Systems

Plant pathogens establish host parasite relationships by entering through infection

such as the spermosphere, rhizosphere and phyllosphere. Hence, protection of sites

vulnerable for the entry and infection of pathogens would offer a better means for

disease management. A combined application of talc based formulation of fluorescent

Pseudomonas comprising of strains Pf 1 and FP 7 through seed treatment, seedling

dip, soil application and foliar spray suppressed rice sheath blight and increased plant

growth better than application of the same strains mixture either through seed,

seedling dip or soil (Nandakumar et al., 2001).The increased efficacy of strain

mixtures through combined application might be due to PGPR formulations 269

increasing the population of fluorescent Pseudomonas in both the rhizosphere and

phyllosphere (Viswanathan and Samiyappan, 1999).

2.5 PGPR Commercial products

Research inventions from China, Russia,and several other western countries

during the early 1950 have proved the potential use of bacteria to be explored for

plant diseases management. Owing to the potential of PGPR, the first commercial

product of Bacillus subtilis was introduced during 1985 for the use of growers by

33

Gustafson, Inc. (Plano, Texas) in US. The strains of B. subtilis A-13, GB03,GB07

were sold for the management of soilborne pathogens under the trade names of

Quantum®, Kodiak® and Epic® respectively (Broadbent et al.,1977). Release of

Bacillus based products during 1985 was resulted in the increase in market size for the

use of bacterial products in crop disease management. Blackman et al., (1998) stated

that 60-75% of the cotton crop in US is treated with B. subtilis for the management of

soilborne pathogens encountered in cotton ecosystem. Among several PGPR strains

Bacillus based products gains momentum for commercialization. Because, Bacillus

spp., produce endospores tolerant to extremes of abiotic environments such as

temperature, pH, pesticides, and fertilizers (Blackman et al., 1998).Owing to the

potentiality of Bacillus spp.,18 different commercial products of Bacillus origin were

sold in China to mitigate soilborne diseases (Blackman et al., 1998). The registered

commercial products of PGPR are listed in Table 2.1 (Nakkeeran et al., 2005).

34

Table 2.1 Commercial products of PGPR in plant disease management

Product Target

pathogens/disease

Crops

recommended

Manufacturer

Bio-Save 10, 11,

100, 110, 1000 TM-

P. syringae ESC-100

Botrytis cinerea,

Penicillium,

Mucor pyroformis,

Geotrichum candidum

Pome fruit (Biosave

100) and Citrus

(Biosave 1000)

Eco Science

Corp,

Produce Systems

Div., Orlando

Blight Ban A506 -

P. fluorescens A 506

Erwinia amylovora and

russet - inducing bacteria

Almond, Apple,

Apricot, Blueberry,

Cherry, Peach,

Pear, Potato, Tomato

Plant Health

Technologies

USA

Cedomon TM -

P. chloroaphis

leaf stripe, net blotch,

Fusarium sp, spot blotch,

leaf

spot and others

Barley and Oats,

potential for wheat and

other cereals

Bio Agri AB,

Sweden

Campanion -

B. subtilis GB03 Rhizoctonia,® Pythium,

Fusarium and

Phytophthora

Horticultural crops

and turf

Growth products,

USA

Conquer TM -

P. fluorescens

P. tolassii Mushrooms

Mauri Foods,

Australia

Victus TM -

P. fluorescens

P. tolassii Mushrooms

Mauri Foods,

Australia

BioJect Spot -less

P. aureofaciens

Dollar spot, Anthracnose,

P. aphanidermatum

Turf and other crops Eco Soil

Systems, San

Diego, CA

Deny -

Burkholderia

cepacia

Rhizoctonia,Pythium,

Fusarium and diseases

caused by lesion, spiral,

lance, sting nematodes.

Alfalfa, Barley, Beans,

Clover, Cotton, Peas,

Sorghum,Vegetable

crops and Wheat

Stine Microbial

Products,

Shawnee, KS

35

Product Target

pathogens/disease

Crops

recommended

Manufacturer

KodiakTM, Kodiak

HBTM, Epic TM,

Concentrate TM,

Quantum 4000 and

System 3 TM -

B. subtilis GB03

Rhizoctonia solani,

Fusarium spp,Alternaria

spp, and Aspergillus spp

Cotton, Legumes Gustafson, Inc.,

Dallas,

USA

Bio Yield -

Combination of

B. subtilis and

B.amyloliquefaciens

Broad spectrum action

against

Greenhouse pathogens

Tomato,Cucumber,

Pepper and Tobacco

Gustafson, Inc.,

Dallas,

USA

Rhizo-Plus -

B. subtilis strain

FZB 24

Against R.

solani,Fusarium.,

Alternaria,Sclerotinia and

Verticillium

Greenhouses grown

crops,forest tree

seedlings,

ornamentals,

and shrubs.

KFZB

Biotechnik

GMBH,

Berlin,Germany

Serenade -

B. subtilis strain

QWT713. Available

as wet table powder

Powdery mildew, Downy

mildew, Cercospora leaf

spot, early blight, late

blight, brown rot, fire

blight , others.

Cucurbits, Grapes,

Hops, Vegetables,

Peanuts, Pome fruits,

stone fruits

and others

AgraQuest, Inc.,

Davis,

USA.

Sonata TM ASO

B. pumilus strain

QST 2808

Fungal pests such as

molds, mildews, blights,

rusts and to

control Oak death

syndrome

Used in nurseries,

landscapes, oak trees

and green house crops

Agra Quest, Inc.,

Davis,

USA

System 3 - Bacillus

subtilis GB03 and

chemical pesticides

Seedling pathogen Barley, Beans, Cotton,

Peanut, Pea, Rice,

Soybean

Helena Chemical

Co.,Memphis

USA

AtEze

P. chlororaphis

strain 63-28

Pythium spp., Rhizoctonia

solani,Fusarium

oxysporum

Ornamentals and

vegetables

EcoSoil Systems,

Inc., San Diego,

CA

36

Amidst these obstacles, since PGPR has its own potentiality in plant disease

and pest management several products have been registered for the practical use of

farming community.Sixty to 75% of cotton crops raised in U.S. are treated with

commercial product of B. subtilis (Kodak®) effective against soilborne pathogens

such as Fusarium and Rhizoctonia. It is also used in peanut, soybean, corn, vegetables

and small grain crops (Blackman et al., 1998). In China, PGPR have been in

commercial development for over than two decades,and are referred as yield

increasing bacteria (YIB).They were applied over an area of 20 million hectares of

different crop plants (Kilian et al.,2000). In India, more than 40 stakeholders from

different provinces have registered for mass production of PGPR with Central

Insecticide Board, Faridabad, Haryana through collaboration with Tamil Nadu

Agricultural University, Coimbatore, India for the technical support and information

(Ramakrishnan et al., 2001).The market size of PGPR usage is increasing constantly

under greenhouse and field conditions, finding solutions for the above obstacles will

create a break through in the adoption of biocontrol agents for field applications.

37

2.6 The Objectives of the Study

In this thesis, six projects are included. They are based on isolation, optimization

and characterization of phosphate solubilizing bacteria from rice rhizosphere

(Paper I), Screening Siderophore Producing Bacteria as Potential Biological Control

Agent for Fungal Rice Pathogens in Thailand (Paper II), Screening and Optimization

of Indole-3-Acetic Acid Production and Phosphate Solubilization from Rhizobacteria

Aimed at Improving Plant Growth (Paper III),Antifungal activity of a novel

compound from Burkholderia gladioli strain SN 4.1 against plant pathogenic fungi,

Biocontrol of Pythium damping-off in Rice by Rhizobacteria and Biocontrol of

Rhizoctonia solani in Bean by Rhizobacteria.