Novel approaches of beneficial Pseudomonas in mitigation of plant diseases – an appraisal

Abstract

Control of plant diseases has always remained a challenge as diseases affecting plant health are a major and chronic threat not only to food production, but also to ecosystem stability worldwide. As agricultural production intensified over the past three decades, producers became dependent on agrochemicals as a relatively reliable method of crop protection. However, growing concerns regarding continued use of agrochemicals, posing adverse effects on human health besides posing the threat of environmental deterioration, has driven search for novel environment friendly methods to control plant diseases that in turn can contribute to the goal of sustainability in agriculture. Mitigation of plant diseases by naturally inhabiting antagonistic micro-organisms such as plant growth-promoting rhizobacteria has gained much importance as biocontrol agents seem to be the best possible measures for saving plants from phytopathogenic organisms without causing any harmful effect to mankind as well as to the environment. Mechanisms of microbial antagonism toward phytopathogenic organisms include competition for nutrients and space, production of siderophores, hydrogen cyanide, antibiotics, and/or production of fungal cell wall-degrading lytic enzymes. The present review is aimed at exploring benefits of natural alternatives for agrochemicals along with the study of their antagonistic mechanisms that makes them a novel substitute to agrochemicals.

Keywords:

• agrochemicals
• antagonism
• biocontrol
• plant growth-promoting rhizobacteria (PGPR)
• siderophores

Introduction

World's population is increasing at an alarming rate and is expected to increase up to 7.5 billion by 2025 (Sharma et al. 2001). Around 70% of the total population lives in the rural areas of developing countries, where poverty, food insecurity, and nutritional deficiency are major problems encountered in day-to-day life. Low productivity in agriculture is considered to be a major cause of poverty, food insecurity, and poor nutrition, especially in countries where agriculture is the driving force for broad economic growth and poverty alleviation. One of the major challenges for twenty-first century is ecologically sound, compatible strategies in agriculture for sustainable crop production as emerging and re-emerging plant diseases continuously challenge our ability to safeguard plant growth worldwide. Under such circumstances, control of plant diseases is considered to be an important need for increased agricultural productivity.

Diseases constitute a major setback to low crop production worldwide as they affect plants right from the planting stage upto harvesting and storage of the produce (Amusa 2006). Conventional control techniques involving use of agrochemicals are not only ineffective in providing long-term control, but also have detrimental effects on humans as they have the property to persist by getting accumulated at various steps of the food chain, besides exposing beneficial soil microbial community to unrealistic concentrations of agrochemicals (Botelho and Mendonça-Hagler 2006). In short, continued chemical treatment of crop plants has necessitated the need to reduce increased usage of agrochemicals with increased emphasis on the implementation of natural means of controlling pests and diseases. Keeping in mind ill-facets associated with conventional techniques, technological advances in implementing natural means of pests and diseases control are needed to minimize unanticipated environmental impacts besides causing increased crop productivity to fulfill the needs of ever-increasing world population.

With change in paradigm from chemical era to environmental era, knowledge of the molecular basis of plant–microbe interactions has become important need for sustainable crop protection based on the use of beneficial micro-organisms. Biological control refers to, ‘purposeful utilization of specific micro-organisms, introduced or naturally inhabiting different ecological niches that interfere with plant pathogens and pests, in a nature-friendly ecological approach to overcome the problems caused by standard chemicals in a way towards protection of plants’ (Haas and Defago 2005). Plants constitute an excellent ecosystem for micro-organisms, therefore from plant perspective antagonistic micro-organisms form an important group of plant-associated micro-organisms that are involved in plant growth promotion, in eliciting physical and chemical changes related to plant defense besides acting as elicitors of tolerance to heavy metals and abiotic stresses, such as drought, salt, and nutrient deficiency. At present, a variety of biological controls are available, but further development and effective adoption requires a greater understanding of the complex interactions of these micro-organisms with plants, people, and environment.

Rhizosphere, root colonization, and nutrient acquisition

Through the ages, course of evolution has been seen to show coexistence between plants and micro-organisms with interactions ranging from mutualism to antagonism. Competitive colonization of rhizosphere and successful establishment in the root zone is considered as prerequisite for effective biocontrol regardless of the mechanism(s) involved (Weller 1988). Traits that promote colonization of rhizosphere include motility, attachment, and efficient nutrient supply. It is well known that increase in the number of attachment structures correlates with the efficiency of adherence and root colonization. Attachment of Pseudomonas cells to plant roots is believed to occur through pilli, cell surface proteins, and polysaccharides (Duijff et al. 1997). O-antigen side chain of outer membrane lipopolysaccharides have been found to be associated with adhesion of cells benefiting root colonization. In certain species, due to impaired synthesis of O-antigen, there occurs reduction in ability to colonize roots (Howie et al. 1987; Rainey 1999). Mutants of Pseudomonas fluorescens lacking flagella have been reported to be inefficient in colonizing potato roots (Schorth and Hancock 1981). Similarly, non-motile mutants of Pseudomonas chloraphis were many fold impaired in tomato tip colonization. Due to this inability, they have lost ability to fight infections in spite of their ability to produce extracellular antifungal metabolites (DeWeger et al. 1987; Lynch 1990; Marschner 1995).

Microbial activity in the rhizosphere plays key role in suppressing soil-borne plant diseases as roots are very prone for the entry of pathogens in both natural and managed agricultural soils (Hariprasad and Umesha 2007; Rani et al. 2007). Roots exude high molecular weight compounds like sugars, amino acids, organic acids, phenolics, fatty acids, and enzymes into the rhizosphere. Organic acids and sugars act as carbon source while as amino acids act as a source of nitrogen. Root exudes alter both physical as well as chemical characteristics of soil that in turn contribute in making soil a suitable habitat for microbial community to flourish. Rhizosphere competence of biocontrol agents comprises of effective root colonization along with the ability of biocontrol agents to survive and proliferate over the considerable period of time in presence of indigenous microfloral population..

Rhizobacteria in relation to plant disease suppression

All soils have a natural level of disease suppressive activities (Baker and Cook 1974; Cook 2000). Long-term management of soils either reduces or increases this level of suppression. Natural suppressiveness is associated with the physical properties of soils and is relatively independent of crop history while as induced suppressiveness is wholly dependent on agricultural practices (Schorth and Hancock 1981). In contrary to phyllosphere, where micro-organisms are subjected to various environmental stresses like strong fluctuations in humidity, temperature, and light, rhizosphere is more protected and as such a permanent habitat to micro-organisms. Several microbes such as Azotobactor sp., Bacillus subtilis, fluorescent Pseudomonads, and Rhizobium sp. in recent times have became forerunners of biocontrol agents against various phytopathogens (Tuzun 2001). Among these rhizosphere competent bacteria, fluorescent Pseudomonads have emerged as the largest and potentially most preferred candidates for biocontrol of plant diseases due of their catabolic versatility, excellent root colonizing ability, and their capacity to produce a wide range of enzymes and metabolites that favor the plant to withstand various biotic as well as abiotic stresses (Anith et al. 1999; Ramamurthy et al. 2001; Mayak et al. 2004; Vivekananthan et al. 2004). Fluorescent Pseudomonads suppress fungal pathogens either directly through the production of various secondary metabolites or indirectly by inducing plant-mediated defense reactions like scavenging iron in the rhizosphere environment through the release of siderophores (O'Sullivan and O'Gara 1992; Loper and Henkels 2000; Braun and Braun 2002). Species of Pseudomonas are also known to excrete various lytic enzymes (chitinases and β-1,3-glucanases), which digest the fungal cell wall chitin and glucan so as to use them as source of carbon and energy (Gooday 1990; Leah et al. 1991; Garbeva et al. 2004). Although, production of extracellular lytic enzymes is quite common antagonistic mechanism, it however does not contribute to antagonism in all cases (Dunne et al. 1998; Sharifi-Tehrani et al. 1998; Adesina et al. 2007). In short, mechanisms of microbial antagonism toward plant pathogenic fungi include production of siderophores, hydrogen cyanide (HCN), antibiotics, and/or production of fungal cell wall-degrading lytic enzymes.

Siderophore-mediated suppression

Dominance of gram negative bacteria, in particular Pseudomonas sp., in the rhizosphere has been frequently reported (Lugtenberg and Dekkers 1999; Sigler et al. 2001). Iron, one of the essential elements required for the growth of almost all the organisms expect a few like lactobacilli devoid of heme proteins, is the fourth most abundant element in the earth's crust (Archibald 1983; Neilands 1995). In spite of its abundance, there occurs scarcity of bioavailable iron in soil habitats as it occurs largely in insoluble (Fe^{2 +}) form, i.e. unavailable for direct microbial assimilation (Crichton and Charloteaux-Wauters 1987; O'Sullivan and O'Gara 1992). Ferric (Fe^{3 +}) form of iron is present in soil in a very small amount (∼10^–18 M at pH 7.4), too low to support growth of micro-organisms having need of iron in the concentration of 10^–6 M. Under such circumstances, micro-organisms secrete iron-binding ligands called siderophores having ability to make ferric form of iron available to host micro-organism (Whipps 2001).

Siderophores are extracellular, low molecular weight microbial compounds which actively sequester iron with high affinity and transfer it to the interior environment of cell. Most siderophores are water soluble and as such are secreted in to exterior environment. Siderophore-producing Pseudomonads colonizing roots of several crops including cereals, pulses, oilseed, and vegetables have significantly enhanced their yield by restricting growth of deleterious bacteria and fungi (Elad and Baker 1985; Neilands and Leong 1986; Loper and Buyer 1991). Soil Pseudomonads produce yellow green water soluble siderophores, grouped on structural basis into hydroxamate and phenolate type structures, having different affinities to capture ferric form of iron from the surrounding environment (Lemanceau et al. 1992; Neilands 1995). Generally, fluorescent Pseudomonads produce pseudobactin or pyoverdin siderophores that possess greater ability to chelate iron available in soil and make it unavailable to pathogen as a result of which pose a serious threat to the survival of pathogenic micro-organisms (Alabouvette et al. 2006). Transport of ferric (Fe^{3 +}) form of iron from exterior into interior of an organism occurs when ferric form of iron combines with pyoverdin to form ferripyoverdin that interacts with specific outer membrane receptors of the producer of the pigment. Analysis of pyoverdin or pseudobactin type of siderophores differs among various fluorescent Pseudomonads in terms of composition, number, and configuration of amino acids in the peptide backbone.

In spite of the ability of siderophores to acquire iron from insoluble hydroxides or from iron adsorbed to solid surfaces, siderophores also extracts iron from iron complexes, such as ferric citrate, ferric phosphate, Fe-transferin, ferritin, or from iron bound to sugars, glycosides, or even from iron chelators like EDTA by ferric/ligand exchange reactions (Lovley et al. 1997). A myriad of environmental factors, such as concentration and the form of iron, degree of aeration, adequate supply of carbon, nitrogen, and phosphorus, pH, and light in addition to presence of trace elements such as magnesium, zinc, and molybdenum has been found to modulate the siderophore production. Although various bacterial siderophores differ in their abilities to sequester iron, but in general they all deprive pathogenic fungi of this essential element as fungal siderophores have lower affinity for iron.

Hydrogen cyanide (HCN)-mediated suppression

Cyanide is a secondary metabolite produced by some gram negative bacteria, such as P. fluorescens, Pseudomonas aeruginosa, and Chromobacterium violaceum (Castric 1975; Askeland and Morrison 1983). P. fluorescens CHA0 is an aerobic, root-colonizing biocontrol bacterium that protects several plants from root diseases caused by soil-borne fungi especially by the production of HCN (Voisard et al. 1994). HCN production by strain CHA0 contributes to the suppression of a disease caused by Thielaviopsis basicola. Production of HCN favored by iron sufficiency in waterlogged, oxygen depleted soils is a major factor that contributes to the antagonism of P. fluorescens CHA0 against soil-borne tobacco pathogen T. basicola, causing black root rot disease of tobacco (Keel et al. 1989; Blumer and Haas 2000; Tarnawski et al. 2006). It is assumed that HCN acts directly on pathogen without causing any damage to the plant.

HCN is formed stoichiometrically from glycine in an oxidative reaction catalyzed by a membrane bound enzyme, HCN synthase. Several models have been proposed for bacterial HCN synthesis. However, as nucleotide sequence of three HCN synthase subunits shows similarities with known dehydrogenases, it therefore supports the dehydrogenase model (Castric 1977; Blumer and Haas 2000). According to dehydrogenases model, glycine is first oxidized to iminoacetic acid [H-C(NH)-COOH], followed by splitting of C–C bond with a concomitant second dehydrogenase reaction resulting in the production of HCN and CO₂ (Wissing 1974). In the three HCN synthase subunits, HcnA shows similarity with a clostridial formate dehydrogenase, where as HcnB and HcnC show similarity with amino acid dehydrogenases (oxidases). Based on sequence comparison with biochemically characterized amino acid oxidases, it is predicted that HCN synthase is a flavoenzyme (Olsiewski et al. 1980; Chlumsky et al. 1995). Hence, flavin adenine dinucleotide (FAD) acts as a stimulator of this reaction whereas pyrrolnitrin (PRN, an inhibitor of flavin enzymes) inhibits the reaction. Cyanogenesis is induced by oxygen-limiting conditions as HCN synthase is very sensitive to oxygen but abolished by iron depletion (Castric 1994). Synthesis of HCN is induced by high ferric iron concentrations whereas conditions of low iron are inhibitory (Weisbeek and Gerrits 1999). In addition to this, an iron chelator strongly inhibits cyanide formation in in vitro conditions.

Antibiotic-mediated suppression

Combination of efficacy, speed of action, ease of use, and low cost of the synthetic insecticides (organochlorines, organophosphates, carbamates, pyrethroids, etc.) drove many botanicals to near obscurity in most industrialized countries. Several years after synthetic insecticides were firmly entrenched in agricultural production, suspected issues of widespread environmental contamination, toxicity to non-target organisms and most importantly negative effects on human health had led to resurgence of interest in ‘natural’ means of pest control, which as a whole has intensified searches for new sources of botanical insecticides (Isman 2008). At the end of 2001, there were approximately 195 registered biopesticide active ingredients and 780 products (http://www.epa.gov/pesticides/biopesticides). At present, many products based on biological control agents are available (Table 1). However, commercial development of products must follow several criteria: safety, production costs, large application on major crops along with effectiveness against broad range of target organisms. Hence, successful biocontrol of plant pathogens requires not only a better understanding of the complex regulation of antimicrobial and/or antifungal metabolite production by antagonists in response to environmental factors, but also a comprehensive picture of dynamic composition of bacterial rhizosphere communities that triggers efficient root colonization.

Table 1. Commercial biocontrol products available for use against plant pathogens.

Product	Biocontrol organism(s)	Applicable to crops	Target pathogen/disease	Manufacturer
AtEze	Pseudomonas chlororaphis 63-28	Ornamentals and vegetables grown in greenhouses	Wilt diseases as well as stem and root rots	Turf Science Laboratories
Bio-save 10LP, 110	Pseudomonas syringae	Pome fruit, citrus, cherries, and potatoes	Botrytis cinerea, Penicillium spp., Mucor pyroformis, Geotrichum candidum	JET Harvest Solutions
Frostban A, B, C, D	Pseudomonas fluorescens strains A506 and 1629RS, Pseudomonas syringae 742RS	Certain fruits, almond, potato, and tomato crops	Frost-forming bacteria	Frost Technology Corporation
Spot-Less	Pseudomonas aureofaciens Tx-1	Turf grass	Dollar spot, anthracnose, pythium, pink snow mold	Turf Science Laboratories
Biocontrol organism other than Pseudomonas
Companion	Bacillus subtilis GB03, other B. subtilis, B. lichenformis, B. Megaterium	Greenhouse and nursery	Rhizoctonia, Pythium, Fusarium, and Phytophthora	Growth Products, Ltd.
EcoGuard	Bacillus licheniformis SB3086	Turf grass	Dollar spot	Novozymes Biologicals, Inc.
Kodiak	Bacillus subtilis GB03	Cotton, legumes	Rhizoctonia solani, Fusarium sp., Alternaria sp., and Aspergillus sp. that attack roots	Gustafson, Inc.
YieldShield	Bacullus pumillus GB34	Soybean	Soil-borne fungal pathogens causing root diseases	Gustafson, Inc.
Mycostop	Streptomyces griseoviridis strain K61	Field, ornamental, and vegetable crops	Fusarium sp., Alternaria brassicola, Phomopsis sp., Botrytis sp., Pythium sp., and Phytophthora sp. that cause seed, root, and stem rot along with wilt disease	AgBio Inc.
Source: APS Biological Control Committee (http://www.oardc.ohio-state.edu/apsbcc) (Raudales and McSpadden Gardener 2008).

In the light of above circumstances, an important aspect of antagonism of plant growth-promoting rhizobacteria (PGPR) is production of secondary antimicrobial and/or antifungal metabolites. Production of antimicrobial and/or antifungal metabolites is subjected to complex regulation, allowing bacteria to sense their own population density, and to respond different environmental stimuli (Lugtenberg et al. 1991; Bloemberg and Lugtenberg 2001). Antagonistic micro-organisms like fluorescent Pseudomonads produces a wide range of different antimicrobial and/or antifungal secondary metabolites, such as 2,4-diacetylphloroglucinol (DAPG), PRN, pyoluteorin (PLT), phenazines (PHZs), and cyclic lipopeptides (Cook et al. 1995; Raaijmakers et al. 2002; Picard et al. 2004; Weller 2007). Genes responsible for the synthesis of these antibiotics are highly conserved among various groups of bacteria. However, concentration at which these compounds are toxic toward pathogenic bacteria, fungi, and nematodes depends on the compound and the target. In fungal pathogens, they may affect the electron transport chain (PRN, PHZ), membrane integrity (biosurfactants), or cell membrane and zoospores (DAPG) (Haas and Defago 2005; Raaijmakers et al. 2006).

2,4-Diacetylphloroglucinol (DAPG)

DAPG is a phenolic antibiotic produced by many fluorescent Pseudomonads exhibiting antifungal, antibacterial, and phytotoxic activities (Reddi et al. 1969; Nowak-Thompson et al. 1994; Bangera and Thomashow 1996). DAPG is a polyketide, synthesized by condensation of three acetyl-CoA molecules with one molecule of malonyl-CoA, resulting in the production of a precursor monoacetylphloroglucinol (MAPG), which after subsequent transacetylation generates the final product, DAPG.

DAPG is believed to be a major determinant in the biological control of plant diseases by Pseudomonas sp., such as take-all disease of wheat caused by Gaeumannomyces graminis var. tritici (Ggt) and black root rot disease of tobacco caused by T. basicola (Weller and Cook 1983; Keel et al. 1989; Harrison et al. 1993; Raaijmakers and Weller 1998). DAPG production involves a set of six genes, including phl D, phl B, phl C, phl A, phl E (transcribed in same direction) and phl F, an repressor exhibiting helix-turn-helix DNA-binding motif regulating the Phl operon (Cook et al. 1995; Bangera and Thomashow 1996). The phl D gene is an essential gene involved in the biosynthesis of DAPG. DAPG induces its own biosynthesis. However, its production is affected by a large number of abiotic factors, such as Fe^{3 +}, Zn^{2 +}, Cu^{2 +}, Mo^{2 +}, and glucose. Fe^{3 +} and sucrose have been reported to increase levels of both DAPG and MAPG in P. fluorescens F113, where as in P. fluorescens Pf-5 and CHA0 its production is reported to be stimulated by glucose (Duffy and Defago 1999). Extracellular metabolites of plant pathogens such as fusaric acid produced by Fusarium oxysporium sp. suppress the expression of genes responsible for the biosynthesis of DAPG as is reported for P. fluorescens CHA0 (Duffy and Defago 1997).

Pyrrolnitrin (PRN)

PRN, a chlorinated phenylpyrrole, is a broad spectrum antifungal metabolite having its history backdated. Arima et al. (1964) described it for the first time as antifungal substance produced by Burkholderia pyrrocina. However, its production has also been reported from P. fluorescens, Pseudomonas chlororaphis, Pseudomonas aureofaciens, Burkholderia cepacia, and Myxococcus fulvus. Synthesis of PRN occurs from aromatic amino acid, tryptophan, in a multistep process mediated by various enzymes encoded by prn A, prn B, prn C, and prn D genes of PRN operon (Hamill et al. 1970). Product of prn A gene catalyzes the chlorination of l-tryptophan to 7-chlorotryptophan followed by its conversion to aminopyrrolnitrin (APRN) via, monodechloroamino pyrrolnitrin (MDA) – byproduct of prn B and prn C genes while as product of prn D gene catalyzes the final step of oxidation of APRN to PRN. PRN operon is regulated by a global regulator gene, gac A (gac is an acronym for global activator of antibiotic and cyanide synthesis) (Duffy and Defago 2000; Haas and Keel 2003). Howell and Stipanovic (1979) reported PRN as an effective metabolite produced by the soil-inhabiting P. fluorescens responsible for imparting protection to cotton seedlings against infection by Rhizoctonia solani. Tazawa et al. (2000) reported that PRN produced by P. fluorescens is associated with decline in takeoff disease of wheat.

Pyoluteorin (PLT)

PLT, a polyketide antibiotic, consists of a resorcinol ring linked to bichlorinated pyrrole moiety. It is produced by several strains of Pseudomonas. It has been found to be associated with the suppression of a series of plant diseases caused by pathogenic fungi, besides possessing bactericidal and herbicidal properties as well. Biosynthesis of PLT occurs from amino acid proline in a series of coupled condensation reactions followed by chlorination and oxidation at different stages of the multistep reaction catalyzed by at least 10 enzymes encoded by 10 genes (plt L, M, R, A, B, C, D, E, F, G) of PLT operon (Kraus and Lopez 1995; Nowak-Thompson et al. 1997). Among them, plt B and plt C encode Type I polyketide synthetase; plt A, plt D, and plt H encode three halogenases; and plt G encodes thioesterase. PLT operon is regulated by plt R gene, which acts as transcriptional activator of the operon. It shows resemblance with Lys R family of transcriptional factors. Production of PLT is mainly associated with the inhibition of oomycete class of fungus including Phythium ultimum causing Phythium-damping disease (Nowak-Thompson et al. 1999).

Phenazines (PHZ)

Natural PHZs include more than 50 pigmented heterocyclic nitrogen-containing secondary metabolites synthesized by members of Pseudomonas sp. (Laursen and Nielsen 2004). Fluorescent Pseudomonad members including strains of P. fluorescens, P. chlororaphis, and P. aeruginosa are best known PHZ producers. P. aeruginosa, a common soil inhabitant and opportunistic human pathogen, is the first PHZ-producing micro-organism reported to produce characteristic blue–green pigment pyocyanin (PYO, 5-N-methyl-1-hydroxyphenazine). Besides producing PYO, it also produce several other PHZs like 1-hydroxy-phenazine, phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), aeruginosin A (5-methyl-7- amino-1-carboxymethylphenazinium betaine), and aeruginosin B (5-methyl-7-amino-1- carboxy-3-sulfophenazinium betaine) (Holliman 1969; Byng et al. 1979). Absorption spectra of PHZs include two distinct maxima in the UV range and at least one in the visible range that varies according to the nature and position of substituents on the heterocyclic ring (Mavrodi et al. 2006).

Shikimic acid is considered as the basic precursor for the synthesis of PHZ and its derivatives. Mavrodi et al. (2001) have reported that a set of seven genes (phz A, B, C, D, E, F, G) in two copies are responsible for the synthesis of PCA in the genome of P. aeruginosa PA01. Among them, phz C encodes 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase, catalyzing the first step of shikimate pathway, besides acting to redirect intermediates from primary metabolism into PHZ biosynthesis. The gene phz F encoding protein required for the production of PCA is assisted by two other proteins of ∼163 amino acids for protein stability while as product of phz G gene is required for PCN synthesis. Besides the above mentioned set of seven structural genes, PHZ operon also encodes some additional genes involved either in transcriptional regulation or in PHZ modification such as phz H gene encoding an aminotransferase responsible for the conversion of PCA to PCN (a green PHZ compound characteristic of P. chlororaphis) (Chin-A-Woeng et al. 1998).

Thomashow and Weller (1988) identified a PHZ antibiotic, PCA as a biocontrol factor produced by P. fluorescens 2-79. This strain, originally isolated from the rhizosphere of wheat, was found to suppress take-all disease of wheat caused by the fungal pathogen Ggt. PYO, PCA, and other PHZs are typical secondary metabolites produced by bacteria in particular Pseudomonads that benefit producer organism by contributing to its ecological fitness and competitiveness in natural habitats such as in the rhizosphere.

In addition to above mentioned antimicrobials and/or antifungal agents, Pseudomonads especially fluorescent ones also produce different types of cyclic lipopeptides (polymers of 9–11 amino acids in the peptide ring with fatty acid molecule at one end of amino acids) (Nielsen et al. 2002). Synthesis of cyclic lipopeptides is catalyzed by peptide synthetase without involvement of ribosomes. They are having tendency to reduce surface and interfacial tensions besides having ability to inhibit microbial growth. Rhamnolipids, viscoisinamide, and tensine are the common cyclic lipopeptides, produced by strains of soil-inhabiting Pseudomonas, and are capable of lysing zoospores of oomycetes besides inhibiting mycelial growth of fungi (Nielsen et al. 1999, 2000; Kim et al. 2000). Amphisin is a new member of dual-functioning compounds like tensin and viscosinamide that have both biosurfactant and antifungal properties. Besides the above mentioned cyclic lipopeptides produced by members of Pseudomonas group, members of the Bacillus group have also been reported to produce bioactive compounds like lipopeptide surfactins (e.g. surfactin) and iturins (e.g. iturin A, iturin D), which possess both biosurfactant and antimicrobial properties. Surfactin and iturin compounds are cyclic lipoheptapeptides containing a β-hydroxy fatty acid and a β-amino fatty acid as lipophilic components.

Lytic enzymes and plant disease suppression

Plant beneficial micro-organisms are known to antagonize phytopathogens through competition for niches or nutrients and parasitism that involve production of hydrolytic enzymes (chitinase, glucanase, protease, and cellulose, capable of lysing fungal cell wall or degradation of chlamydospores, oospores, conidia, sporangia, and zoospores. Adams (1990) has referred this process of physical destruction of the fungal cell wall by the action of hydrolytic enzymes produced by biocontrol agents as mycoparasitism.

In plant-beneficial Pseudomonas sp., production of a number of degradative enzymes is believed to be involved in the biocontrol process against various fungal agents. Antifungal enzymes, such as chitinases, β-1,3-glucanase etc., produced by Trichoderma sp. and by many strains of Pseudomonas sp. can profitably be exploited in combination with other antifungal antibiotics to achieve biocontrol of such plant pathogenic diseases by causing destruction of chitin and β-1,3-glucan that constitutes major structural components of plant pathogenic fungi (Heeb and Haas 2001; Zhang et al. 2001; Fogliano et al. 2002). It is reported that extracellular chitinase and laminarinase synthesized by Pseudomonas stutzeri digest and lyse mycelia of Fusarium solani (Lim et al. 1991).

Induced resistance as a mechanism of plant disease suppression

In addition to indirect biocontrol activity of rhizosphere-inhabiting micro-organisms, several others have a direct positive effect on plant growth through activation of plant defense mechanisms (Pieterse et al. 2002; Chisholm et al. 2006). However, duration of this response is critical as it reflects difference between coping and succumbing to biotic challenge of various plant pathogens. Defense mechanisms triggered by a stimulus prior to infection reduce chances toward acquiring disease. Induced resistance is a state of enhanced defensive capacity developed by a plant in response to an appropriate signal. It is often one of several modes of action by which micro-organisms can benefit plant health. Plants have evolved a number of inducible defense mechanisms against pathogen attack; but generally two types of induced resistance are reported: systemic acquired resistance (SAR) and induced systemic resistance (ISR).

SAR is a mechanism that confers long-lasting protection against a series of plant pathogenic micro-organisms. It is activated in many plant species by pathogens that cause necrosis, either as part of the hypersensitive response (characterized by rapid cell death at the site of infection) or as a symptom of disease. The resistance conferred is long-lasting, sometimes for the lifetime of plant and effective against a broad-spectrum of pathogens including viruses, bacteria, fungi, and oomycetes (Hammond-Kosack and Jones 1996; Ryals et al. 1996; Sticher et al. 1997). SAR requires salicylic acid (SA) as signal molecule associated with accumulation of pathogenesis-related (PR) proteins thought to contribute resistance against various disease-causing agents. In case of Arabidopsis, it is reported that in response to SA, a positive regulator protein, NPR1 interacts with TGA transcription factor at the site of nucleus thereby resulting in the activation of SAR (Pieterse et al. 1998; Spoel et al. 2003). NPR1 is also required for induced resistance pathways, induced by nonpathogenic bacterium P. fluorescens, where it mediates cross-talk of SA signaling pathway with jasmonic acid (JA) and ethylene (C₂H₄) signaling pathways, which as a whole confers resistance to insects as well as to other necrotrophic pathogens.

Like SAR, rhizobacteria-mediated ISR triggers different signaling pathway, independent of SA and PR gene inductions as reported from Arabidopsis thaliana, where signaling pathway is mediated by JA and ethylene signaling (Pieterse et al. 1996). ISR does not confer complete protection, but it does protect plant from various types of root pathogens without requiring direct interaction between the resistance-inducing micro-organisms and the pathogen (van Loon et al. 1998; Zehnder et al. 2001). Several cell surface constituents of biocontrol bacteria, such as lipopolysaccharide, flagella etc., trigger ISR. ISR is also acquired by plants following exposure to compounds produced by plant-beneficial bacteria, e.g. volatile 2,3-butanediol (Ryu et al. 2004), siderophore pyoverdine (Maurhofer et al. 1994), DAPG (Iavicoli et al. 2003), and cyclic lipopeptide surfactants (Ongena et al. 2007; Tran et al. 2007).

Ethylene, the only gaseous phytohormone produced by almost all plants, is known as the ‘stress or wounding hormone’ as its production in plant is induced in response to various physical or chemical perturbations caused by osmotic stress (characterized by drought, low temperature, and high-salt concentration) and heavy metals (Salisbury 1994; Saleem et al. 2007). Ethylene is synthesized from methionine via, S-adenosyl-l-methionine (AdoMet) and cyclic non-protein amino acid 1-aminocyclopropane-1-carboxylate (ACC) through involvement of two enzymes: one ACC synthase catalyzing conversion of AdoMet to ACC and other ACC oxidase catalyzing conversion of ACC to ethylene. Among its myriad of effects on plant growth and development, ethylene production can cause inhibition of root growth. Glick (1995) put forward a theory indicating mode of action of some PGPR through production of ACC deaminase, an enzyme having capacity to cleave ACC (immediate precursor in the biosynthetic pathway for ethylene in plants) into a-ketobutyrate and ammonia. ACC deaminase (a pyridoxal-5-phospahate-dependent polymeric enzyme) has been reported to be secreted by many species of bacteria and fungi, but was first studied in a soil bacterium Pseudomonas sp. (Wang et al. 2002; Yang et al. 2009). Previous studies with an PGPR, Paenibacillus polymyxa associated with A. thaliana, Achromobacter piechaudii ARV8 associated with pepper (Capsicum annum L.), and tomato (Solanum lycopersicum L.) have been found to enhance drought tolerance in them through production of ACC deaminase (Mayak et al. 2004). Inoculation of plants with PGPR containing ACC deaminase activity is helpful in sustaining plant growth under stress conditions by reducing ethylene production in the roots of host plants that ultimately result in root lengthening.

Discussion

Excessive use and misuse of agrochemicals, as well as fear-mongering of pesticides, have led to considerable change in people's attitude toward use of agrochemicals in agriculture. Almost every country is struggling to arrive at an effective regulatory regime as continuous use of agrochemicals holds a double-edged sword of rendering pests resistant besides leading to ecosystem collapse. Agriculture sector confronted by many stresses (biotic as well as abiotic), calls for the efforts toward biotechnological revolution that will give a promising solution for sustainable environmental friendly agriculture. Importance of PGPR in agriculture is on the rise as they hold a promise of suppressing various diseases, as a means toward sustainable strategies for management of soil-borne pathogens. Maximizing the potential for successfully developing and deploying biocontrol agents, biocontrol begins with a carefully crafted microbial screening procedure, proceeds with developing mass production protocols that optimize product quantity and quality and at the end devising a product formulation that preserves shelf-life, aids product delivery and enhances bioactivity. Tremendous progress has been made in recent years to identify effective multitasking biocontrol agent active against broad range of pathogens to overcome inconsistent effects of agrochemicals. Among various biocontrol agents, Pseudomonads are intended to play an active role by controlling the activity and viability of plant pathogenic fungi. Their biocontrol activity is directly correlated with production of a series of antibiotics, such as PRN, DAPG, PLT, PHZs, and other lytic enzymes. Potential for using strains of beneficial Pseudomonas particularly fluorescent Pseudomonads as biocontrol agents has been demonstrated on many crops. Comprehensive study of the dynamic microbial consortia could shed light on the process to select successful strains promoting plant growth through suppression of plant diseases. Despite evidences regarding role of fluorescent Pseudomonads in curbing plant diseases under laboratory and green house conditions, results in the field are less consistent. Because of these restraints, practical requirement for screening of inoculants formulation restricts their reliability in the field, necessary for its development as a reliable commercial component in the management of sustainable agricultural systems.

Knowledge of the biological environment in which such biocontrol agent will be used and the study of the mechanism which they employs to produce a stable formulation is critical for successful biocontrol as lack of knowledge often contributes to the downfall of a biocontrol agent. Advanced knowledge on plant protection properties of fluorescent Pseudomonad antagonists such as rhizosphere competence, genes contributing to rhizosphere competence and suppressing various diseases along with factors affecting effective root colonization are needed for the management of soil-borne pathogens. Although, sequences of genetic loci responsible for biosynthesis of antibiotics are well characterized, new ventures for modulation of their biocontrol efficiency need an in depth analysis of the interactions between the fluorescent Pseudomonads and the surrounding environment. With the help of recombinant DNA technology, different manipulations would unfold many other mechanisms that can be applied to such bacterial strains for improvisations of their qualities by creating transgenic strains that would combine multiple mechanisms of action for the destruction of various pathogens.

Conflict of interest

Authors declare that they have no conflict of interest.

Acknowledgements

One of the authors, Arif Tasleem Jan would like to thank Council for Scientific and Industrial Research, India, for SRF.