1. Introduction

1.1 Plant growth-promoting rhizobacteria 

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In the rhizosphere, that is the portion of soil on the plant root or its close vicinity, bacteria are abundantly present, most often organized in microcolonies (Bloemberg et al. 2001). The plant rhizosphere is an essential soil ecological environment for plant–microorganism interactions, which include colonization by a variety of microorganisms in and around the roots that may result in symbiotic, endophytic, associative, or parasitic relationships within the plant, depending on the type of microorganisms, soil nutrient status, and soil environment (Albareda et al. 2006). In this sphere, intensive interactions are taking place between the plant, soil, soil microfauna and microorganisms, where bacteria are the most abundant microorganisms (Antoun and Kloepper, 2001). The region around the root is relatively rich in nutrients because as much as 40% of plant photosynthates are lost from the roots, hence it supports the large microbial population (Ping et al. 2004).

The activity and diversity of microorganisms adjacent to roots differs from the activity and diversity of the microorganisms in the bulk soil (Wang et al. 2005). In the bulk soil population sizes were larger, but in the rhizosphere the phylogenetic diversity is more restricted (Marilley & Aragno, 1999; Berg et al. 2005). The high concentration of easily metabolizable organic compounds in the rhizosphere sustain microbial populations that are more active, denser but less diverse than those present in bulk soil (Inbar et al. 2005).

Rhizobacteria are rhiszosphere competent bacteria that colonize and proliferate on all the ecological niches found on the plant roots at all stages of plant growth, in the presence of a competing microflora (Antoun and Kloepper, 2001). Based on their effects on the plant, microbes interacting with plants can be categorized as pathogenic, saprophytic and beneficial. Pathogens can attack leaves, stems or roots. Microbes in their interactions with plants, no matter whether the microbe is beneficial or pathogenic, often use the same mechanisms, although in different combinations and for different purposes. Similarly, it is obvious that microbes in their interaction with plants use similar strategies as in their interactions with other eukaryotes such as fungi and humans (Lugtenberg et al. 2002). Some of these rhizobacteria not only benefit from the nutrients secreted by the plant root but also beneficially influence the plant in a direct or indirect way, resulting in a stimulation of its growth (Bloemberg et al. 2001).

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Plant growth-promoting rhizobacteria (PGPR), first defined by Joseph W. Kloepper and Milton N. Schroth, include a wide range soil bacteria that colonize the roots of plants following inoculation onto seed and enhance plant growth by increasing seed emergence, plant weight, and crop yields (Ping et al. 2004 and Ryu et al. 2004). Besides colonizing the root surfaces and the closely adhering soil interface PGPR can also enter root interior and establish endophytic populations. Many of them are able to transcend the endodermis barrier, crossing from the root cortex to the vascular system, and subsequently thrive as endophytes in stem, leaves, tubers, and other organs. The extent of endophytic colonization of host plant organs and tissues reflects the ability of bacteria to selectively adapt to these specific ecological niches. Consequently, intimate associations between bacteria and host plants can be formed without harming the plant. Although, it is generally assumed that many bacterial endophyte communities are the product of a colonizing process initiated in the root zone, they may also originate from other source than the rhizosphere, such as the phyllosphere, the anthosphere, or the spermosphere (Compant et al. 2005).

PGPRs have drawn much attention in recent years because of their contribution to the biological control of plant pathogens and the improvement of plant growth. Inoculation of plants with ‘dual’ microbial inoculants, or even a consortium of them, is becoming more important in a framework of sustainable agriculture for the advantage their beneficial effects afford, providing there is no competition between inoculants (Albareda et al. 2006). Extensive research has demonstrated that PGPRs could have an important role in agriculture and horticulture in improving crop productivity. In addition, these organisms are also useful in forestry and environmental restoration. As agricultural production intensified over the past few decades, producers became more and more dependent on agrochemicals as a relatively reliable method of crop protection helping with economic stability of their operations. However, increasing use of chemical inputs causes several negative effects, i.e., development of pathogen resistance to the applied agents and their nontarget environmental impacts. Furthermore, the growing cost of pesticides, particularly in less-affluent regions of the world, and consumer demand for pesticide-free food has led to a search for substitutes for these products. There are also a number of fastidious diseases for which chemical solutions are few, ineffective, or nonexistent. PGPR is thus being considered as an alternative or a supplemental way of reducing to the use of chemicals in agriculture in many different applications (Lucy et al. 2004 and Compant et al. 2005).

1.1.1 Mechanisms of plant growth-promoting rhizobacteria

Diverse mechanisms are involved in plant-bacteria interactions, and in many cases individual PGPR have several mechanisms on their activities to promote the plant growth at various times during the life cycle of the plant (Glick et al. 1999; Berg et al. 2002 and Müller, 2009). Based on their mode of action, PGPRs are grouped into two large classes, namely the PGPRs that directly affect plant metabolism resulting in increased plant growth, seed emergence or improved crop yields and the Biocontrol-PGPRs, which suppress plant pathogens, thereby benefiting the plant indirectly (Ping and Boland, 2004; and Wang, 2005).

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In all mode of action of PGPR, the ability to colonize plant habitats especially roots is important for all successful plant–microbe interactions, which in turn determine inoculum efficacy both for crop yield enhancement and for disease control. This has led to an emphasis on selection of plant-beneficial bacteria that are rhizosphere competent (i.e., beneficial bacteria that effectively colonize the root system) (Kamilova et al. 2005 and Compant2 et al, 2005). Steps of colonization include recognition, adherence, invasion (only endophytes and pathogens), colonization and growth, and several strategies to establish interactions. Plant roots begin crosstalk with soil microbes by generating signals that are recognized by the microbes, which in turn produce signals that initiate colonization (Bais et al. 2006). In Ps e udomonas fluorescens WCS365 the major traits involved in competitive root tip colonization are motility; adhesion to the root; a high growth rate in root exudate; synthesis of amino acids, uracil, and vitamin B1; the presence of the O-antigenic side chain of lipopolysaccharide; the two-component ColR/ColS sensory system; fine-tuning of the putrescine uptake system (the mutant had an impaired pot operon); the site-specific recombinase Sss or XerC; the nuo operon (the mutant had a defective NADH:ubiquinone oxidoreductase); the secB gene involved in a protein secretion pathway; and the type three secretion system (TTSS) (Lugtenberg et al. 2001).

1.1.1.1 Direct plant growth promotion

Even though the molecular basis for the interactions is not always well known, several basic principles of molecular interplay between the PGPRs and plants have been successfully unraveled. The most prominent example is nitrogen fixation by bacteria such as Rhizobium and Bradyrhizobium that can form nodules on roots of leguminous plants such as soybean, pea, peanut, and alfalfa, and convert N2 into ammonia, which in contrast to N2 can be used by the plant as a nitrogen source. The symbiosis between rhizobia and its legume host plants is an important example for plant growth-promoting rhizobacteria (PGPR). The symbiosis is initiated by the formation of root or stem nodules in response to the presence of the bacterium. Lipooligosacharide signal molecules that are secreted by the bacterium play a crucial role in this process. The bacteria penetrate the cortex, induce root nodules, multiply and subsequently differentiate into bacteroids, which produce the nitrogenase enzyme complex. Within the root nodules, the plant creates a low oxygen concentration, which allows bacterial nitrogenase to convert atmospheric nitrogen into ammonia. In return, the plant supplies the bacteria with a carbon source. The molecular interaction between the plants (providing the carbon source) and the microorganisms (providing the nitrogen supply) is highly complex and involves many factors (Freiberg et al. 1997; Bloemberg and Lugtenberg 2001; Berg 2009; and Lugtenberg and Kamilova 2009). However, several bacteria belonging to the genus Azospirillum, Burkholderia , and Stenotrophomonas have the ability to fix nitrogen as a free living organism (Dobbelare et al. 2003).

Some bacteria are able to influence the hormonal balance in the plant. For example, Pseudomonas putida GR12-12 and Enterobacter cloacae UW4 contain the gene for ACC (1-aminocyclopropane-1-carboxylate) deaminase, which can cleave the plant ethylene precursor ACC, and thereby lower the level of ethylene in a developing or stressed plant (Hall et al. 1996 and Hontzeas et al. 2004). PGPR that contain the enzyme ACC deaminase, when bound to the seed coat of a developing seedling, provides a mechanism for ensuring that the ethylene level does not become elevated to the point where root growth is impaired. With the longer roots, survival of some seedlings will be enhanced especially during the first few days after the seeds are planted. Similarly, ACC deaminase-containing bacteria bound to the roots of plants can act as a sink for ACC and protect stressed plants from some of the deleterious effects of stress ethylene (Glick 2005). Several other forms of stress are relieved by ACC deaminase producers, for example effects of phytopathogenic bacteria, and resistance to stress from polyaromatic hydrocarbons, from heavy metals such as Ca2+ and Ni2+, and from salt and draught (Glick et al. 2007).

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Mineral supply is also involved in plant growth promotion and low levels of soluble phosphate can limit the growth of plants. Some plant-growth promoting bacteria solubilize insoluble phosphate from either organic or inorganic bound phosphates which makes phosphorous available to the plants (Rodriguez and Fraga, 1999). Pseudomonas fluorescens NJ-101, Pseudomonas fluorescens EM85 and Bacillus amyloliquefaciens FZB45 are to name of some bacteria that have ability to solubilize insoluble phosphate, therefore enhance nutrient availability to plants and facilitating plant growth (Idris et al. 2002; Bano and Mussarat. 2004; Dey et al. 2004 and Vassilev et al. 2006). Idris et al. concluded that phytase activity of B. amyloliquefaciens FZB45 is important for plant growth stimulation under phosphate limitation. Extracellular pyhtase activity is mainly produced during the late stage of exponential growth and during the transition to stationary growth phase, suggesting that similar to other extracellular depolymerases phytase acts as a `scavenger' enzyme after exhaustion of rapidly metabolized nutrient sources (Idris, et al. 2002). Another mineral that is important for the plant growth is iron. The shortage of bioavailable iron in soil habitats and on plant surfaces generates an intense competition among microorganisms. By far, the most common mechanism of iron acquisition by microorganisms involves chelation of ferric iron by siderophores. Under iron-limiting conditions PGPR produce low-molecular-weight compounds called siderophores to competitively acquire ferric ion. The release of siderophores chelates iron and makes it available to the plant root (Loper and Henkels. 1997; Ping and Boland. 2004; and Katiyar and Goel. 2004)

Phytohormones are involved in the control of growth and in almost every important developmental process in plants. Many PGPR can produce phytohormones, such as auxins, cytokinins, and gibberellins (Salamone et al. 2001; Ortiz-Castro et al. 2008 and Joo et al. 2009). Indeed, three types of plant growth promoting substances have been detected in the supernatant of Azospirillum cultures, these are auxins, cytokinins and gibberellines. Of these, the auxin IAA (indole-3-acetic acid) is quantitatively the most important one as it can directly benefit the plant root system by promoting the development of lateral roots and apical meristem divisions that lead to lengthening of the roots (Malhotra and Srivastava 2009). Experiments with Azospirillum mutants altered in IAA production prove the view that after Azospirillum inoculation IAA causes increased rooting, which in turn enhances mineral uptake (Steenhoudt and Vanderleyden 2000). Idris et al. (2007) proved that biosynthesis of IAA in B. amyloliquefaciens FZB42 affected its ability to promote plant growth. By inactivating the genes involved in tryptophan biosynthesis and in a putative tryptophan-dependent IAA biosynthesis pathway, IAA concentration and plant growth promoting activity in respective mutants were reduced. Gibberellins are plant hormones which control several different physiological processes such as the stimulation of stem elongation by stimulating cell division and elongation, the stimulation of bolting/flowering in response to long days, the break of seed dormancy in some plantswhich require stratification or light to induce germination, the stimulation of enzyme production (α-amylase) in germinating cereal grains for mobilization of seed reserves, the induction of maleness in dioecious flowers (sex expression), the inducement of a parthenocarpic (seedless) fruit development, and the retardation of senescence in leaves and citrus fruits (Joo et al. 2004). Production of gibberellins which promote plant growth has been reported in different bacteria such as Rhizobium Phaseoli, Acetobacter diazotrophicus, Herbaspirillum seropedicae, B. pumilus, B. licheniformis and B. macroides (Joo et al. 2005; Atzhorn et al.1988; Bastian et al. 1998 and Gutierrez-Manero et al. 2001). Cytokinins are a class of phytohormones produced by plants and microorganisms which may play an essential role in regulating cytokinesis, growth and development in plants (Aloni et al. 2006). Hence, it can be expected that plant inoculation with PGPR capable of producing cytokinins may increase the level of cytokinins in root tissues which in turn may have an impact on plant growth (Ortis-Castro et al. 2008). Cytokinins are thought to be the signals involved in mediating of environmental stress from roots to shoots (Jackson, 1993).

Some rhizobacteria, such as strains from B. subtilis, B. amyloliquefaciens, and Enterobacter cloacae, promote plant growth and induce systemic resistance by releasing volatile organic compound (VOC) (Ryu et al. 2003 and Ryu et al. 2004). Analysis of the volatiles emitted from Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, revealed that two compounds, 3-hydroxy-2-butanone (acetoin) and 2,3-butanediol, were shared by both bacterial strains whereas other PGPR strains that did not trigger enhanced growth via volatile emissions also did not share this same subset of volatile components. Other components of the complex bouquet from B. subtilis (e.g. decane, undecane, undecane-2-one, tridecan-2-one and tridecan-2-ol) were not active. Furthermore, pharmacological applications of 2,3-butanediol enhanced plant growth whereas bacterial mutants blocked in 2,3-butanediol and acetoin synthesis were inactive in plant growth promotion (Ryu et al. 2003). VOC can also trigger the growth of the plant by regulating auxin homeostatis in which the gene expression for auxin production was upregulated. In addition, microarray data revealed coordinated regulation of cell wall loosening enzymes that implicated cell expansion with B. subtilis GB03 exposure (Zang et al. 2007).

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The cofactor Pyrroloquinoline quinone (PQQ) is recently regarded as a plant growth promotion factor produced by Pseudomonas fluorescens B16 (Choi et al. 2007). In mammals, pyrroloquinoline quinone (PQQ) functions as a potent growth factor, although its biological functions are not fully understood (Steinberg et al. 1994). Mutations in pqq genes abolished plant growth-promotion activity of wild-type B16, whereas synthetic PQQ promotes growth of tomato and cucumber plants. This study provides evidence that PQQ is a plant growth-promotion factor because of its antioxidant activity. However, it cannot be excluded that the effect is indirect because PQQ is a cofactor of several enzymes, e.g., involved in antifungal activity and induction of systemic resistance (Choi et al. 2007).

1.1.1.2 Indirect plant growth promotion

Pathogenic microorganisms which damage the plant health are a major and chronic threat to food production and ecosystem stability worldwide. Over the past few decades, producers became more dependent on agrochemicals as a relatively reliable method of crop protection in order to intensify their agricultural production. However, increasing use of chemical inputs causes several negative effects, i.e., development of pathogen resistance to the applied agents and their nontarget environmental impacts as well as negative impact to human health (Gerhadson 2002; Leach and Mumford 2008). The use of microbes as form of biological control to manage diseases is an environment-friendly approach. These biocontrol agents are a natural enemy of the pathogen, and if they produce secondary metabolites, they do only locally, on or near the plant surface, i.e., the site where they should act (Lugtenberg and Kamilova 2009). Such microorganisms can produce substances that may limit the damage caused by phytopathogens, e.g. by producing antibiotics, siderophores, and a variety of enzymes and can also function as competitor of pathogens for colonization of sites and nutrients (Timmusk 2003). Some PGPR strain can also lead to a state of induced systemic resistance (ISR) in the treated plant. ISR occurs when the plant’s defense mechanisms are triggered and primed to resist infection by pathogens (Van Loon, 1998). Schematic illustration of some important mechanism of biological control of plant diseases by bacteria is shown in Fig. 1.

Figure 1.  Illustration of the most important mechanisms of biological control of plant diseases by bacteria

In all cases illustrated here, biocontrol begins by coating seeds with the biocontrol bacterium. (a) Antibiosis. The bacterium colonizes the growing root system and delivers antibiotic molecules around the root, thereby harming pathogens that approach the root (indicated by stars). (b) Induced systemic resistance (ISR). Local root colonization is sufficient to induce ISR. Many bacterial products induce systemic signaling, which can result in protection of the whole plant against diseases caused by different organisms. The latter aspect of ISR resembles innate immunity in humans and animals. (c) Competition for nutrients and niches. Biocontrol bacteria acting through this mechanism excel in fast chemotactic movement along the growing root in their efficient hunt for root exudate components, thereby outcompeting the pathogen in scavenging nutrients and in occupying niches on the root (Lugtenberg and Kamilova 2009).

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PGPR can produce a variety of antibiotics including 2,4-diacetylphloroglucinol (DAPG), phenazines, hydrogen cyanide, pyrrolnitrin, pyoluteorin, viscosinamide and tensin produced by pseudomonads (Nielsen et al. 1999; Nielsen et al. 2000; Bloemberg and Lugtenberg 2001; Haas and Defago 2005) and zwittermycin A, kanosamine, bacillomycin D and fengycin produced by Bacillus ( Raaijmakers et al. 2002; Koumoutsi et al. 2004). Biocontrol agents from P. fluorescens act rather nonspecific in their ability to protect plants from soil phytopathogens. Indeed, each strain can typically work in more than one pathosystem, i.e. protect more than one plant species from often distinct pathogens, provided the rhizosphere is successfully colonized. They have been mostly studied for protection of crop plants from phytopathogenic oomycetes and fungi and to a lesser extent bacteria and nematodes (Couillerot et al. 2009). The production of anti-fungal metabolites (AFMs) in Pseudomonas involves a complex regulation. Main factors in the regulation of the biosynthesis of most AFMs are global regulation and quorum sensing. Global regulation is directed by the gacS/gacA genes, which encode a two-component regulatory system that senses an as yet unknown signal(s). Quorum sensing involves the production of N-acyl homoserine lactone (AHL) signal molecules by an AHL synthase such as LuxI. The AHL then binds to and activates a transcriptional regulator, such as LuxR. The activated form of the transcriptional regulator then stimulates gene expression (Bloemberg and Lugtenberg 2001). An antibiotic produced by Bacillus cereus and Bacillus thuringiensis, zwittermycin A, adversely affects the growth and activity of a wide range of microorganisms, including several plant pathogenic fungi and in particular Phytophthora and Pythium species (Raaijmakers et al. 2002).

Various PGPR can reduce the activity of pathogenic microorganisms not only through microbial antagonisms, but also by inducing a state of systemic resistance in plants, which provides protection against a broad spectrum of phytopathogenic organisms including fungi, bacteria and viruses. This enhanced defensive capacity is termed induced systemic resistance (ISR) (Van loon, 2007). The mechanisms of ISR include (1) developmental—escape: linked to growth promotion, (2) physiological—tolerance: reduced symptom expression, (3) environmental: associated with microbial antagonisms in the rhizosphere, and (4) biochemical—resistance: induction of cell wall reinforcement, induction of phytoalexins and pathogenesis-related proteins, and “priming” of defense responses (Berg 2009). The evidence of the ISR was first described by Van Peer et al. (1991) in carnation that was systemically protected against Fusarium oxysporum f.sp. dianthi upon treatment with strain Pseudomonas fluorescens WCS417 and by Wei et al. 2001 on cucumber (Cucumis sativus) with reduced susceptibility to foliar disease caused by Colletotrichum orbiculare. Before challenge inoculation, no increase in phytoalexin levels could be detected in induced and uninduced plants but, upon subsequent inoculation with F. oxysporum f.sp. dianthi, phytoalexin levels in ISR-expressing plants rose significantly faster than in uninduced plants. Bacillus pumilus SE34 induces ISR in bean (Phaseolus vulgaris) against the root-rot fungus F. oxysporum f.sp. pisi. by appositions containing large amounts of callose and phenolic materials, thereby effectively preventing fungal ingress (Benhamou et al. 1996). Studies on mechanisms show that elicitation of ISR in Bacillus spp is associated with ultrastructural changes in plants during pathogen attack and with cytochemical alterations (Kloepper et al. 2004). ISR acts through a different signaling pathway to that regulating systemic acquired resistance (SAR), the ISR pathway is induced when the plant is challenged by non-pathogenic organism. Bacterial determinants that are responsible to trigger ISRs include siderophores, the O-antigen of lipopolysacharide, N-acyl-homoserine lactones, salicylic acid and VOCs (e.g., 2,3-butandiol).  Whereas some PGPR activate defense-related gene expression, other examples appear to act solely through priming of effective resistance mechanisms, as reflected by earlier and stronger defense reaction once infection occurs (Bloemberg and Lugtenberg 2001; Conrath et al. 2002, Berg 2009). Investigations of the signal transduction pathways of elicited plants suggest that Bacillus spp. activate some of the same pathways as Pseudomonas spp. Pseudomonad PGPR that trigger ISR is dependent on JA, ethylene, and Npr1, a regulatory gene that encodes salicylate dehydrogenase, but independent of SA, a result that is in agreement with several strain of Bacillus spp. However, in other cases, ISR elicited by Bacillus spp. is dependent on salicylic acid and independent of jasmonic acid and NPR1. The VOCs of Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a that trigger ISR involved signal transduction pathways that were independent of SA, JA, and Npr1. In addition, in some cases ISR by Bacillus spp leads to accumulation of the defense gene PR1 in plants, ISR by Pseudomonas spp. does not. (Kloepper et al. 2004; Ryu et al. 2004).

Competition for niche and nutrients can also be a fundamental mechanism by which PGPB protect plants from phytopathogens. In the rhizosphere there are various suitable nutrient-rich niches as a result of exudation of compounds attracting a great diversity of microorganisms, including phytopathogens (Compant et al. 2005). Known chemical attractants present in root exudates include organic acids, amino acids, and specific sugars (Welbaun et al. 2004). Some exudates can also be effective as antimicrobial agents and thus give ecological niche advantage to organisms that have adequate enzymatic machinery to detoxify them. This implies that PGPR competence highly depends either on their abilities to take advantage of a specific environment or on their abilities to adapt to changing conditions (Bais et al. 2004). Competition may concern the acquisition of organic substrates released by seeds and roots as well as micronutrients such as soluble iron, which is often in limiting amounts in soil. Iron acquisition entails the production of iron transporters (siderophores), noticeably fluorescent pyoverdines (Couillerot et al. 2009). Although various bacterial siderophores differ in their abilities to sequester iron, in general, they deprive pathogenic fungi of this essential element since the fungal siderophores have lower affinity (Loper et al. 1999).

1.2 Transposons mutagenesis

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Transposons are mobile genetic elements that can move from one site to another in the genome with the aid of a recombinase called a transposase. They are ubiquitous and present in Eubacteria, Archaea, and Eukarya, including in humans in which they constitute a significant fraction of the genome. Transposons are widely used as tools for random mutagenesis in vitro and in vivo in a variety of organisms ranging from gram-negative Escherichia coli to eukaryotes, and engineered transposons have been developed that incorporate a variety of useful features (Bordi et al. 2008; Petzke and Luzhetskyy 2009). Transposable elements are the causative agents of various insertion, deletion, inversion and chromosomal fusion mutations. When inserted in the appropriate location of the genome, mutation caused by transposons can inactivate or activate critical genes (Chandler and Mahillon 2002; Reznikoff 2003). Transposable elements in bacteria range from simple insertion sequence (IS) elements that consist of a gene(s) for transposition bounded by inverted repeat sequences, to composite transposons composed of a pair of IS elements that bracket additional genetic information for antibiotic resistance or other properties, to more complex conjugative transposons that exhibit hybrid properties of transposons, plasmids, and bacteriophages (Hayes, 2003). Numerous transposon delivery systems have been developed for Escherichia coli and other gram negative bacteria. However, in many cases these incorporate selectable markers that are not conducive to their use in gram-positive bacteria (Bordi et al. 2008). There are two mechanisms in transposon movement, namely ‘cut and paste’ and ‘replicative transposition’. In cut and paste mechanism the element is excised from its resident location and inserted at a new position, whereas in replicative transposition, the transposition process involves cointegration of the donor replicon that harbors the transposon and the target molecule with concomitant duplication of the transposon (Hayes 2003).

The transposons most favored as genetic tools are those that insert randomly or near-randomly, or can be manipulated to behave in this way. Tn917 transposon, a streptococcal Tn3-like transposon, was the first transposon developed for use in B. subtilis. It was adapted by the incorporation of a promoterless lacZ gene, and the resulting Tn917lac transposon was used to generate large numbers of reporter fusions. Despite their wide use, Tn917 has significant shortfalls in which ninety-nine percent of all Tn917 insertions occur at several “hot-spot” regions of the B. subtilis chromosome (Youngman et al. 1983; Youngmann et al. 1985). More recently, Tn10 and mariner transposon were used for in vivo transposition in B. subtilis. Tn10, a transposon isolated from E. coli, was adapted for B. subtilis by fusion of the transposase gene to expression signals appropriate for this bacterium (Petit et al. 1990). Unlike Tn917, Tn10 does not appear to have preferred insertion sites in the B. subtilis chromosome; but it is known to have a strong preference for a 6-bp target sequence. Hence reduces the number of potential Tn10 insertion sites on the B. subtilis chromosome and, as a consequence, Tn10’s effectiveness as a tool for random mutagenesis (Halling and Kleckner 1982; Breton et al. 2006).

Mariner-family transposable elements are a diverse and taxonomically widespread group of transposons occurring throughout the animal kingdom and especially prevalent in insects. Their wide distribution results from their ability to be disseminated among hosts by horizontal transmission and also by their ability to persist in genomes through multiple speciation events (Robertson, 1993; Hartl et al. 1997). Among hundreds of different mariners that have been detected, only two are known to be active. The first is Mos1 which was discovered from Drosophila mauritiana. The second is the Himar1 which was isolated from the horn fly Haematobia irritans. A transposon based on the eukaryotic mariner family of transposons has been used for eubacteria, archaebacteria, and eukaryotic cells (Lampe et al. 1999; Julian and Fehd 2003). Compared to other transposons that have been engineered to construct insertional mutagenesis in bacteria, mariner elements offer several advantages. First, they do not require species-specific host factors for efficient transposition. Second, apart from the dinucleotide TA, mariner elements have no specific sequence requirements for their insertions. Third, they transpose in both eukaryotes and prokaryotes. In addition, transformation with mariner elements usually leads to 10-fold-more mutants than transformation with the Tn917 (Louvel et al. 2005; Picardeau 2010). Due to its effectiveness in transposition, numerous transposon systems based on the mariner transposon family have been applied for mutagenesis in bacteria (Bourhy et al. 2005; Wu et al. 2006; Liu et al. 2007; Kritisch et al. 2008).

1.3  Bacillus amyloliquefaciens FZB42

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Among various group of plant-associated microorganisms, strains of Bacillus have gained more attention as they have several advantages over other biocontrol bacteria in that they are easy to cultivate and store. In addition, they offer a biological solution to the formulation problem due to their ability to form heat- and desiccation-resistant spores, which can be formulated readily into stable products. Hence they can be applied as spores on plant seeds or in inoculants (Reva et al., 2004; Emmert & Handelsmann, 1999). The genus Bacillus is characterised by gram positive, rod shaped, facultative aerobe, endospore forming bacteria that live in soil and often colonise the plant rhizosphere. It has a broad host range, ability to produce different kind of antibiotics and other secondary metabolites important for plant growth (Gardener, 2004).

B. amyloliquefaciens FZB42 is regarded as PGPR due to its biocontrol and phytostimulator activity. Its genome has been sequenced and mapped; therefore it is possible to detect the genes responsible for its plant growth activity (Chen et al. 2007). Phytase activity and auxin production of B. amyloliquefaciens FZB42 which are important for plant growth promotion have been reported (Idriss et al., 2002; Idriss et al. 2007). FZB42 genome analysis revealed the presence of numerous gene clusters involved in synthesis of non-ribosomally synthesized cyclic lipopeptides and polyketides with distinguished antimicrobial action (Chen et al. 2009a; Chen et al. 2009b). For example production of non-ribosomally synthesized peptides such as b acillomycin D and fengycin are able to inhibit growth of phytopathogenic fungi such as Fusarium oxysporum in synergistic way (Koumoutsi et al., 2004). Whereas polyketide compounds such as difficidin and bacilysin act efficiently against fire blight disease caused by Erwinia amylovora (Chen et al. 2009c).

1.4 Aims of the project

B. amyloliquefaciens FZB42 is known as plant growth promoting bacterium due to production of a vast array of secondary metabolites which protect and support the growth of the plant. Several mechanisms of its activities have been reported recently (Idriss, et al. 2007; Koumoutsi et al., 2004; Chen et al. 2009c), however, still many mechanisms are not fully understood.

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The complete genome sequence of B. amyloliquefaciens FZB42 showed that many regions in this genome were still obscure (Chen et al, 2007); hence it needs to be exploited further in order to reveal the unexpected potential for developing agrobiotechnological agents with predictable features. In doing so, transposon mutagenesis will be applied to discover genes that are potentially involved in its plant growth-promoting activity. The mariner-based transposon TnYLB-1 was selected, since it “jumps” into the B. subtilis chromosome with high frequency and requires only a “TA” dinucleotide as the essential target in the recipient DNA. Therefore, it can insert nearly random in all regions of the Bacillus chromosome (Le Breton et al. 2006).

Screening of a mutant library generated by TnYLB-1 transposon will be done to identify the genes involved in rhizosphere competence (swarming ability and biofilm formation) as well as in plant growth-promoting activity. In addition, colonization of B. amyloliquefaciens FZB42 and its mutants on the roots of the plant will be monitored using SEM and CLSM to find out whether or not there is different pattern of colonization. The use of transposon mutagenesis will also be applied to discover novel secondary metabolites by screening the transposon library for mutants impaired in synthesis of antibiotics and in nematocidal activity.


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