REVIEW OF LITERATURE

CHAPTER 2

REVIEW OF LITERATURE

2.1. Genus Pseudomonas

Psezldomonas is an enormously diverse genus of y-Proteobacteria (Galli et al.

1992). This genus consists of ubiquitous saprophytic members of plant, animal and

human pathogens. Psezldornorzas are Gram negative, motile, and oxidase positive.

Members of the genus Pseudornor.zas have very simple nutritional requirements and grow

well under normal conditions in mixed populations with other types of microorganisms

(Foster 1988). Den Dooren deJong (1 926) first characterized Pseudomonas strains

phenotypically on the basis of their nutritional features. Stanier et al. (1966) conducted a

fundamental study on the Pseudomor~as that result in an extensive phenotypic

characterization in which the genus was subdivided into species and species groups.

Conclusions of this study were supported by numerical analysis (Sneath et al. 198 1) and

DNA-DNA hybridization (Palleroni and Doudoroff 1 972). Palleroni et al. (1 973)

conducted rRNA-DNA hybridizations that revealed five rRNA homology clusters (rRNA

groups).

2.2. rRNA groups of Pseudomonas

2.2.1. rRNA group I

The largest rRNA group consists mostly of saprophytic bacteria (P. .flzlorescens,

P. putida, P. clzlororaphis) or pathogenic for humans (P. aerugirzosa), plants (P. cichorii,

P. marginalis, P. syri~zgae, P. sa~~rrstarzoi), mushroo~ns (P. agarici, P. toluasii) and P.

stutzeui, P. nzendocina, P. alculigerzcs and P. pseudoalculigenes. Taxonomically the

fluorescent organisms such as P. uer-zrginosa and P. .fluorescerzs are remarkably

heterogeneous species (Doudoroff and Palleroni 1974; Palleroni 1 992).

2.2.2. rRNA group I1

The second RNA group is called the Pseudomallei-cepacia group. It contains

group of pathogenic species, with exception of P. pichettii (Raltson et al. 1973). The most

remarkable species is P. cepacia, which is a plant pathogen and also a significant human

oppol-tunistic pathogen (Ederer and Matsen 1972). This group also contains P. marginata

(P. glaioli), P, caryophylli, P. pseudornallei, P. mallei and P. solanaceavt~m.

2.2.3. rRNA group I11

Third rRNA group is represented by five species. Two of the species P.

(Comamonas) acidovorans and P. (Comamonas) testosteroni have been shown to be so

distantly related to other Pseudomonas sp. that a new genus, Comamonas has been

proposed (De Vos et al. 1985). The other three phytopathogenic species are P. u11e17ae, P.

vubrilirzea~zs, P. kol2j~zci.). These groups are phenotypically quite different from one

another.

2.2.4. rRNA group IV

Group IV coinprises P. di~nirztlta and P. vcsicularis. These two strains stand as out

group and did not show affinity with any other Psez~domonus (Ballard et al. 1968).

2.2.5. rRNA group V

The fifth rRNA hoinology group constitutes P. multophiliu (now

Stenotvophomonas ~naltophilia) (Palleroni and Bradbury 1993) together with

Xanthomonas species. P. lnultophilia can be found in many natural habitats living as a

saprophyte, and it is also frequently isolated fiom clinical specimens (Palleroni et al.

1973).

A number of Psewdomonus species have not yet been assigned to RNA hoinology

groups. The marine species, facultative autotrophs the poly-P-hydroxyl butyrate utilizing

pseudomonads are among them.

2.3. Fluorescent Pseudowzonas

In colnlnon with the other species of the genus Pseudomolzus, the fluorescent

pseudoinonads are Gram-negative, strictly aerobic, polar flagellated rods. Fluorescent

pseudoinonads are quite heterogeneous and there are no phenotypic traits common to all

members of the group. All fluorescent pseudotnonads fall into one of the five rRNA

group (Palleroni et al. 1973). The Guanine-plus-Cytosine (G+C) content of their DNA

ranges from 58 to 68 mol% (Palleroni 1975).

2.3.1. Fluorescent Pseudomonas as biocontrol and plant growth

promoting agents

Many of the chemicals that are used to control diseases are hazardous to animals

and humans, beneficial organisms, and persist in natural ecosystems. In recent years,

fluorescent pseudoinonad bacteria have been identified as potent biocontrol agents

against phytopathogens.

Saprophytic fluorescent pseudolnonads are typical inhabitants of agricultural field

soils and plant rhizosphere, and are involved in several interactions with plants (Schroth

et al. 1992). In recent years, fluorescent pseudolnonads are considered as biologically

important coinponents of agricultural soils that are suppressive to diseases caused by

pathogenic fungi on crop plants. Fluorescent pseudoinonads suppress disease severities

caused by plant pathogens and enhance growth of a variety of crops like rice (Mew and

Rosales 1986; Sakthivel and Gnanamanickaln 1987), wheat (Weller and Cook 1983),

potato (BUIT et al. 1978; Kloepper et al. 1980) sugar beet (Suslow and Schroth 1982),

radish (Kloepper and Schroth 1978), cotton (Howell and Stipanovic 1980) and cassava

(Hernandez et al. 1986). A number of different fluorescent pseudomonad species such as

P. putida (Scher and Baker 1980), P. aerugi~zosu (Bano and Musal~at 2003; Sunish

kuinar et al. 2005) P. chlorovuplzis (Chin-A-Woeng et al. 1998); P. cepacia (Cattelan et

al. 1999) have been reported as plant growth pronloting rhizobacteria (PGPR) as well as

biocontrol strains against phytopathogenic fungi (de Salmone et al. 2001). List of

antagonistic fluorescent pseudomonad strains, antifungal metabolites and affected

pathogens is presented in Table 1 and 2. However, not all fluorcscent pseudomonads are

antagonistic against pathogens.

Fluorescent pseudomonads are suitable for application as biological control

agents due to their abundant population in natural soils and plant root system (Sands and

Rovira 1971), and their capability to utilize many plant exudates as nutrient (Lungtenberg

et al. 1999). Fluorescent pseudomonads are known to have important traits in bacterial

fitness such as the ability to adhere to soil particles and to the rhizoplane, motility and

prototrophy (de Weger et al. 1994), synthesis of antibiotics (Natsch et al. 1994) and

production of hydrolytic enzymes (Lim et al. 1991; Neilsen et al. 1998; Neilsen and

Sorensen 1999). The antagonistic properties and plant growth promoting abilities of

fluorescent pseudomonads are due to their direct and indirect mechanisms. Indirect

inechanisins used by fluorescent pseudomonads include antibiotic production against

pathogenic bacteria (Thomashow et al. 1990), reduction of iron available to

phytopathogens in the rhizosphere (Scher and Baker 1982), synthesis of fungal cell waIl

degrading enzymes, and conlpetition with detrimental lnicroorganisms for sites on plant

roots. Direct mechanisms of plant growth by fluorescent pseudomonads include the

provision of biologically available phosphorus for plant uptake, sequestration of iron for

plants by siderophores, production of phytohorrnones (Salisbury 1 994), and lowering of

plant ethylene levels (Glick 1995; Glick et al. 1999). In addition, fluorescent

pseudomonads are capable of inducing a systemic resistance against pathogens known as

induced systemic resistance (Van Loon et al. 1 998; Pieterse et al. 200 1 ).

2.4. Mechanisms of plant growth promotion

2.4.1. Solubilization of phosphates by fluorescent pseudomonads

Phosphate solubilizing bacteria are common in the rhizosphere. Secretion of

organic acids and phosphatase are common method of facilitating the conversion of

insoluble fo~ms of phosphorus to plant-available forms (Kim et al. 1998; Richardson

2001). Fluorescent pseudomonad species such as P. chlororuphis, P. pzitidu and P.

aerzlginosa have been identified as phosphate solubilizing rhizobacteria (Cattelan et al.

1999; Bano and Musarat 2003; Sunish ku~nar et al. 2005).

2.4.2. Phytohormones of fluorescent pseudomonads

2.4.2.1. Indole-3-acetic acid (IAA)

The phytohorrnone, indole-3-acetic acid (IAA) is known to be involved in root

initiation, cell division, and cell enlargement (Salisbury 1994). This holmone is

colnmonly produced by rhizobacteria (Barazani and Friedman 1999). IAA-producing

rhizobacteria are believed to increase root growth and root length, resulting in greater

root surface area, which enables the plant to access, Inore nutrients from soil. Patten and

Glick (2002) reported the role of IAA producing P. ptltida in development of the host

plant root system.

2.4.2.2. Cytokinins

Cytokinins are a class of phytohor~nones, which are known to promote cell

divisions, cell enlargement and tissue expansion (Salisbuiy 1994). Cytokinins are

believed to be the signals involved in mediating environmental stress from roots to shoots

(Jackson 1993). Garcia et al. (2001) have reported the production of cytokinins in P.

fluorescens.

2.4.2.3. 1-Aminocyclopropane-1-carboxylate (ACC) deaminase

Ethylene is the only gaseous phytohormone. It is also known as the 'wounding

hormone' because its production in the plant can be induced by physical or chemical

perturbation of plant tissues (Salisbury 1994). Among its myriad of effects on plant

growth and development, ethylene production can cause an inhibition of root growth.

Glick et al. (1998) put forward the theo~y that the mode of action of some plant growth

pronioting rhizobacteria (PGPR) was the production of ACC deaminase, an enzyme that

could cleave ACC, the inllnediate precursor to ethylene in the biosynthetic pathway for

ethylene in plants. ACC dealninase activity would decrease ethylene production in the

roots of host plants and result in root lengthening. Wild type and genetically modified

fluoi.escent pseudomonads were reported as ACC dealninase producers (Glick et al.

1994). Transforming Pseudomonas spp. strains with a cloned ACC dealninase gene

enabled the bacteria to grow on ACC as a sole source of nitrogen and to promote the

elongation of seedling roots (Shah et al. 1998). The growtl~ promotion effects also

expressed in stressful situations such as flooded (Grichko and Glick 2001) or heavy

metal-contaminated soils (Burd et al. 1998; Beli~nov et al. 2001).

2.4.3. Denitrification

Denitrification is a microbial process in which oxidized nitrogen compounds are

used as alternative electron acceptors for energy production when oxygen is limited.

Denitrification consists of four reactions by which nitrate is reduced to dinitrogen by the

inetalloenzymes such as nitrate reductase, nitrite reductase, nitric oxide reductase, and

nitrous oxide reductase. Fluorescent pseudo~nonads are the most comlnon denitrifiers

isolated from temperate soils (Gamble et al. 1977). Fluorescent pseudomonads are able to

adapt to limited oxygen conditions by using nitrogen oxides as alternative electron

acceptors (Stewart 1988), and respiratory nitrate and nitrite reductase have been described

to implicate in the competitiveness of model strains of fluorescent pseudomonad in soil

(Philippot et al. 1995; Glziglione 2000).

2.4.4. Siderophore-mediated iron absorption by fluorescent

pseudomonads

Several species of fluorescent pseudomonads produce siderophores and there is

evidence that a number of plant species can absorb bacterial ~ e ~ ' siderophore complexes

(Becker and Cook 1988; Loper 1988; Bitter et al. 1991). Fluorescent yellow green

siderophores have been named as pyoverdines (PVDs) or pseudobactins (Budzikiewicz

1993, 1997). Besides PVD, P. aevziginosa produces another siderophore called pyoehelin

with a lower affinity for iron (111) (Cox et al. 1981). Fluorescent pseudomonad species

such as P. Jluorescens, P. stutzeri and P, putida produce pseudonlonine (isoxazolidone)

(Lewis et al. 2000; Mossialos et al. 2000; Mercado-Blanco et al. 2001).

2.5. Mechanisms of antagonism

Fluorescent pseudomonads produce an array of secondary metabolites as well as

several cell wall degrading enzynes. Well-characterized secondary metabolites with

biocontrol properties include phenazines, phloroglucinols, pyoluteorin, py~~olnitrin,

lipopeptides, and hydrogen cyanide. These secondary metabolites exert toxic activities

against a wide range of bacteria and fungi.

2.5.1. Secondary metabolites with biocontrol properties

2.5.1.1. Phenazines

Phenazines are intensely coloured N-containing heterocyclic pigments

synthesized by different bacterial strains (Leisinger and Margraff 1979; Budzikiewicz

1993 ; Stevans et al. 1 994). Phenazines exhibit broad-spectrum activity against bacteria

and fungi (Smimov and Kiprianova 1990). Phenazines also play an important role of

microbial competition in rhizosphere, including survival and competence (Mazzola el al.

1 992).

2.5.1.1.1. Phenazine-1-carboxylic acid (PCA)

PCA has been reported froin fluorescent pseudomonads such as P. fluorescens

(Gunrsiddaiah et al. 1986), P. chlororaphis (Pierson and Thamashow 1992) and P.

ner-zrgirzosa (Anjaiah et al. 1998). PCA was dexnonstrated to be effective against various

fungal pathogens such as Gae~c~nuizizor~z~vces g~urniizis var. tr-itici, Pythiz{r?z sp., Polyporus

sp., Rhizoctorziu solurli etc. and bacterial pathogens such as Actirzo/~zyces viscoszls,

Brrcillus subtilis, Etwinia a~zylo~~or-u etc. (Gulusiddaiah et al. 1986; Thomashow et al.

1990).

2.5.1.1.2. Phenazine-1-carboxamide (PCN)

Production of PCN had been reported in fluorescent pseudomonads such as P.

aevugirzosa and P. clzlororaphis (Chin-A-Woeng et al. 1998; Mavrodi et al. 2001a;

Sunish kurnar et al. 2005). PCN differs fi-om PCA with a carboxamide (CONH*) group

replacing the carboxyl (COOH) group at the first position of the phenazine core. PCN is

more stable than PCA and exhibits antihngal activities even in alkaline pH (Chin-A-

Woeng et al. 1998).

2.5.1.1.3. Pyocyanin

The bluish coloured pyocyanin (1-hydroxy-5methyl-phenazine) is predominantly

produced by P. aeruginosa (Demange et al. 1989). The antibiotic cyanomycin, from

Stveptornyces cyanofavus (Funaki et al. 1958) is also known as pyocyanin (Turner and

Messenger 1986). Pyocyanin is toxic to a wide range of Eungi and bacteria (Hassan and

Fridovich 1980).

2.5.1.2. Phloroglucinols

Phloroglucinols are broad-spectrum antibiotics produced by a variety of bacterial

strains. 2,4-diacetyl phloroglucinol (DAPG) is a broad-spectrum phenolic antibiotic

produced by P. Jluorescens Pf-5 (Howell and Stipanovic, 1979), P. Jlt~oresceizs F113

(Fenton et al. 1992), P. Jl~~ovesce~zs CHAO (Keel et al. 1992) and P. .fluor.escens 42-87

(Bangera and Thomashow 1996). This antibiotic also exhibits herbicidal activity

resembling 2, 4-dichlorophenoxyacetic acid (2, 4-D), a cormnonly used post-emergence

herbicide for the control of many annual broad leaf weeds of cereals, sugarcane and

plantation crops (Dwivedi and Johri 2003). DAPG also produces induced systemic

resistance in plants (Dwivedi and Johri 2003) and thus serving as a specific elicitor of

phytoalexins and other similar molecules.

2.5.1.3. Pyrrol compounds

Arima et al. (1964) first reported the broad spectrum antifungal metabolite,

pyrrolnitrin (PRN) (3-chloro-4-(2'-nitro-3'-chlorophenyl) pyrrole). PRN is produced by

fluorescent pseudomonads such as P. fluo?*esce7?s (Kirner et al. 1998) and P. chlororahis

(Elander et al. 1968). Pyrrolnitrin has found its application more as a clinical colnpound

than as an agricultural fungicide. Other variants of pyrrolnitrin (isopyrrolnitrin,

oxypyrrolnitrin and inonodechloropyrronitrin) have lower antifungal activities (Elander et

al. 1968).

2.5.1.4. Polyketide compounds

2.5.1.4.1. Pyoluteorin (PLT)

PLT the chlorinated antifungal metabolite of mixed polyketide/arnino acid origin

produced by certain strains of P,setrdolnonas spp. including soil bacterium, P. .fluorescens

Pf-5 (Maurhofer et al. 1992; Maurhofer et al. 1994; Kraus and Loper 1995; Nowak-

Thompson et al. 1997). Strains producing PLT suppress several soil borne plant diseases

(Howell and Stipanovic 1980; Defago et al. 1990; Maurhofer et al. 1994). Pyoluteoiin is

found to be inore effective against the damping-off disease causing oomycete, P. ultirnt~nz

(Maurho fer et al. 1 992).

2.5.1.4.2. Mupirocin

Mupirocin, the naturally occurring polyketide antibiotic of fluorescent

pseudo~nonads is also known as pseudomonic acid. Mupirocin produced by P.

Jluorescens NCIMB 10586 is highly active against Staphylococcus auveus and a variety

of Gram positive organisms (El-sayed et al. 2003). Mupirocin is also used as a tropical

and intranasal antibiotic (Carcanague 1997).

2.5.1.4.3. 2,3-deepoxy-2,3-didehydrorhizoxin (DDR)

DDR produced by P. chloro~aplzis MA342 is effective against several

phytopathogenic fungi, including net blotch of barley caused by the fungus Dreclzsleva

teres. Y. chlorof*uphis MA342 is con~inercially used in Sweden as a biocontrol agent

under the trade name Cedomon (Tombolini et al. 1999).

2.5.1.5. Peptide antibiotics

Peptide antibiotics are predominately produced in both Gram-positive (Katz and

Demain 1977) and Gram-negative bacteria (Dowling and O'Gara 1994) by a non-

ribosomal multi-enzymatic peptide synthesis (Kleinkauf and Dohen 1990). Recently, it

has been observed that fluorescent pseudo~nonads produce a number of different cyclic

lipopeptides (CLPs) which are usehl in biological control. It is also found that CLP

production is a cormnon trait among fluorescent pseudomonads isolated froin sugar beet

rhizosphere (Nielsen et al. 2002).

Cyclic lipodecapeptide, tensin is produced by P. j7uorescens 96.578 (Nielsen et al.

2000). Tensin showed potent antagonistic activity against the basidiomycete fungus, R.

solani. Significant reduction of R. solani infection was found in sugar beet seeds treated

with tensin producing strain P. .flzforesccns 96.578 mielsen et al. 2000). The mode of

action of tensin on R. solani is still not clearly perceived. However, it is proposed that the

activity might be in synergism with chitinolytic or cell wall degrading enzymes produced

by P. flzioresceizs 96.578 mielsen and Sorensen 1999; Nielsen et al. 2000).

2.5.1 -5.2. Viscosinamide

Viscosinainide is a cyclic lipopeptide produced by P. fl~iorescens DR54 (Nielsen

et al. 1999). This compound shows prominent antihngal and biosurfactant properties

(Nielsen et al. 2000; Thrane et al. 2000; Nielsen et al. 2002). It is highly effective against

R. snlani. In soil conditions, viscosinamide producing P. flzlorescens DR54 is found to

reduce the mycelial biomass and sclerotia formation by R. solani close to the seed or

seedling root surfaces, thus making the fungal biomass inadequate for infection (Thane

et al. 2000).

2.5.1.5.3. Oomycin A

Oomycin A antibiotic produced by P. fluovesce~zs HV37a exhibits suppression of

damping-off disease of cotton caused by P. ultimurn (Gutterson et al. 1988). The genes

afiiE and afuR that encode the enzymes for the synthesis of oomycin have been reported

(Gutterson et al. 1 98 8).

Table 1 List of phenzine and phloroglucinoi producing fluorescent pseudornonads and their

biocontsol ability against different phytopathogenic fungi

Anti fungal Fluorescent Affected pathogen Host plant Reference metabolite pseudoinonad

strain

Phenazine Phenazine- 1 - P. fiuorescevrs cal-boxylic acid 2-79RNlo ( P C 4 PGS12

P. aevuginosa PNA 1

Phenazine-1 - P. chlororuphis casboxamide PCL139 1 (PCN)

P. aeruginosa PUPa3

Pyocyanin P. aertiginosa

Phloroglucinol 2,4-diacetylphl- P. Juorescens oroglucinol Q8rl-96 (DAPG) CHAO

Guezcnzarzrzonz.yces Wheat grmnirzi var. tritici Fzlsarium oxysporuln Corn

G. grarnini var. tritici Wheat

F. o.~v~~porurn Tomato

F. o,uysporzim Chickpea Pythizrrn splendens F. olysporum f. sp. Tomato i-adicisycopevsici

Sarocludiurn ovyzae Rice Rl?izoctonia solani

Septoria tritici Wheat

G. grumini var. t~itici wheat

G. grumini var. tritici Wheat Thielaviopsis basicola, Tobacco P. tiltinzum P. ultimzim Tobacco R. solani Cotton P, ultimzlrn Sugar beet

S. tritici Wheat

Weller 1 983

Georgakopoulos et al. 1994 Pierson and Thomashow 1992 Chin-A-Woeng et al. 1998 Anjaiah et al. 1998, 2003 Chin-A-Woeng et al. 1998

Sunish kumar et al. 2005

Baron et al. 1997 Baron and Rowe 1981 Flaishnan et al. 1990

Raaij~nakers and Weller 2001 Keel et a1.1992

Howell and Stipanovic 1979 S hanahan et al. 1992 Levy et al. 1992

Table 2 List of pyrrol, polyketide, oolnycin and peptide producing fluorescent pseudomonads

and their biocontrol ability against different phytopathogenic fungi

Anti fungal Fluorescent metabolite pseudoxnonad

strain

Affected Host plant Reference pathogen

Pyrrol Pyrrolni trin (PRN)

Polyketide Pyoluteorin (PLT)

Pyoverdin

Pyochelin

Oomycin Oomycin A

Peptide Viscosinamide

P. flz~oresccn.s BL9 1 5 P. cepacia 5.5B

P. juorescens CHAO P. fluorescens 3 5 5 1 P. pzltidu WCS358

P. flzlovescens CHAO P. aeruginosu 7NSK2

R. solurli Cotton R. sokur~i Cotton

P. z~ltimtlnz Cotton R soluni Cotton P. ziltimzrrn Tobacco P. ultirnunz Cotton F. oxysporum Cotton f. sp. raphani P. splendens Tomato P. splendens Toinato

P. ultimum Barley

R. solani Cotton

R. solani Cotton

Ligon et al. 2000 Gal-twright et al. 1995

Howell and Stipanovic 1980 Keel et al. 1992 Loper 1 98 8 Van Wees et al. 1997 Buysens et al. 1994

Gutters011 et al. 1986

Nielsen et al. 1999 Nielsen et al. 2000

2.6. Genetic relation of antibiotic production in fluorescent

pseudomonads

Production of antibiotics in fluorescent pseudomonads is subject to coinplex

regulation. Key factors in the regulation of antibiotics are global regulators and quorum

sensing (Bloemberg and Lugtenberg, 2001). Global regulation is mediated by the gacS

and gacA genes that encode a two-component regulatory system. Quorum sensing

involves the production of N-acyl-hornoserine lactone (AHL) signal lnolecules (Bassler

1999). The gacS and gacA regulatory system also controls quorum sensing, illustrating

the complexity of the regulation of antibiotic production in fluorescent pseudomonads.

The quorum sensing molecule, AHL has been repoi-ted to involve the regulation of

antibiotic production in P. .fluorescens, P. aerzlginosa and P. chlororaphis (Fray et al.

1999; Pessi and Haas 2001). Sigrna factor genes, rpoD and rpoS have been reported to

control production of such antibiotics and enhance the antagonistic activities of

fluorescent pseudomonas (Fujita et al. 1994). In the case of P. flz~oresceizs, a high rpoD

gene dose inight stimulate antibiotic production directly or indirectly. Other form of

global regulation such as Lon protease (Whistler et al. 2000) transcriptional repressor

p1zl.F (Delany et al. 2000) and regulatory (PrrB) RNA (Aarons et al. 2000) on secondary

inet abolite production have also been reported recently.

2.7. Fungal cell wall degrading enzymes

Production of hngal cell wall degrading enzymes excreted by lnicroorganis~ns is

frequently involved in the attack of phytopathogenic fungi (Max-tin and Loper 1999;

Nielsen and Sorensen 1999; Picard et al. 2000). Lysis by cell wall degrading enzymes

excreted by microorganisms is a well known feature of mycoparasitism. Chitinase, P-1,3

glucanase and cellulase are especially important fungus controlling enzymes due to their

ability to degrade the fungal cell wall components such as chitin, P 1,3 glucan and

glucosidic bonds (Potgieter and Alexander 1966; Bartnicki-Garcia and Lipp~nan 1973;

Schroth and Hancock 1981; Chet 1987; Lorito et al. 1996). Chitinase excreting

n~icroorganisins have been reported as efficient biocontrol agents (Sneh 198 1 ; Ordentlich

et al. 1988; Inbar and Chet 1991). Role of chitinase in biological control as well as in

plant defense mechanisms has been docunlented well (Shapira et al. 1989). Nielson et al.

(1998) reported that in the sugar beet rhizosphere fluorescent pseudomonads inhibit plant

pathogenic hngi R. solani by production of cell wall-degrading endochitinase. Biological

control of F. solani, mainly via laminarinase and chitinase activities of P. stutzeri YPL-1

has been reported (Lim et al. 1 99 1). Fridlender et al. (1 993) reported that P- 1,3 glucanase

producing P, cepacia decreased the incidence of diseases caused by R. solar~i, S. rolfsii

and P. wltimurn.

2.8. Taxonomic identification and phylogenetic analyses of fluorescent

pseudomonads

Fluorescent pseudomonads were identified by dichoto~nous keys based on the

most discriminating characters (Jacques 1994; Bossis 2000). In numerical taxonomy,

classifications of strains by mathe~natical analyses are done (Sneath and Sokal 1973).

Highly similar strains are clustered into phenons; bacterial species showed similarity at

80% and 60% similarity cor-respond to genera. Fluorescent pseudo~nonad strains have

been delineated as P. Jluoresce~zs lineage including the species P. flz40veLscens together

with P. chlovoraphis, P. rnar.girzalis, P. tolaasi, rind P. viridzflava on the basis of 16s

rRNA (Moore et al. 1996).

Taxonomic identification based on fatty acid composition and protein profiles are

of most concern. Stead (1 992) identified an rRNA homology group of a P. fluouescens

based on fatty acid analyses. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis

(SDS-PAGE) analyses showed the protein bands that enable the identification of P.

aerzlgirzosa, P. pzltida and P. Jlz~orescens at specific level. On the basis of primary

characteristics and carbon assimilation patterns, Sakthivel and Gnanamanicka~n (1989)

reported the abundance of P. fluorescens biovar 111 in south Indian soils and rice

rhizosphere. Lemanceau et al. (1995) reported the effect of two plant species, flax and

tomato on the diversity of fluorescent pseudoinonads. Most isolates from flax and tomato

belonged to P. putida bv. A and to P. fluorescens bv. 11, respectively.

Diversity of fluorescent pseudomonads of ~nycorrhizosphere has been reported

with the taxonomic identification of 90% of isolates as P. Jltroresccr~s on the basis of

phenotypic traits. Latour et al. (1996) observed that the diversity of fluorescent

pseudornonads was higher in soil than in plant tissue. Rangarajan et al. (2001) observed a

high degree of diversity anlong fluorescent pseudomonad strains isolated from rice

cultivated along a salinity gradient. PCR based analysis has already been successfully

used to differentiate species wi~liin numerous genera, in environmental contexts,

including Psezrdornonas spp. Amplified ribosomal DNA restriction analysis (ARDRA)

has been considered as a convenient method to discriminate among closely related

species, being much faster than DNA-DNA hybridization. Using phenotypic and

genotypic (rep-PCR) markers, Frey et al. (1 997) observed higher intraspecies diversity of

P. j7uorescens in soil, compared to the diversity of Laccavia bicolor on the

mycon-hizoplane.

The diversity of the membrane-bound nitrate reductase (navG) and nitrous oxide

reductase (nod) genes in fluorescent pseudomonads isolated froin soil and rhizosphere

environments was characterized together with that of the 16s rRNA gene by a PCR-

restriction fragment length polymorphism (RFLP) by Delonne et al. (2003). Distribution

in the different ribotypes of strains harboring both the navG and nos2 genes and of strains

missing both genes suggests that these two groups of strains had different evolutionary

histories. Both dissimilatory genes showed a high degree of polymorphism.

The DNA gyrase B subunit (gyr.B) and RNA polymerase sigtna factor 70 (rpoD)-

based phylogenetic classification was applied to the analyses on the intragenetjc

relationships on genus Pseudonzona.~ (Yamarnoto and Harayama 1998; Yarna~noto et al.

2000). Molecular phylogeny deduced fro111 a single locus may be unreliable due to the

stochastic nature of base substitutions or to rare horizontal gene transfer events and

therefore, combined gvrB and ipoD sequence analyses have been used to achieve more

accurate estimate of the phylogeny.

The importance of antagonistic fluorescent pseudomonads in agriculture has

initiated numerous molecular as well as ecological investigations (Rainey et al. 1994;

Lelnanceau et al. 1995; Frey et al. 1997; Johnsen and Nielsen 2 999; Latour et al. 1999).

Characterization of antagonistic fluorescent pseudomonads is important for

understanding their ecological role in agriculture as well as important for the registration,

patenting, recognition and quality checking. Since the taxonomic affiliations of

fluorescent pseudonlonads are a~nbiguous a polyphasic approach is required to clarify

their interspecific position within the genus Psez~domonas.