1. Introduction

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Throughout evolution plants in nature have been subject to constant attack by plant pathogenic microbes, such as fungi and bacteria, as well as by insect pests. As a consequence of this intimate relation between plants and their attackers, a wide range of defence systems against pest and pathogen attacks has evolved in plants (Ryan, 1990; Bowles, 1990; Chessin and Zipf, 1990). Although pathogens and pests have developed systems to overcome these defences, only a very limited number of them succeed in infecting the plants (Collinge et al., 2001). Resistance has, therefore, been continually present in nature. As domestication of plants with partially high productiveness has not been able to preserve natural defence mechanisms, it has not been possible to optimise plant production and yield potential under the influence of biotic and abiotic factors to guarantee quantitative and qualitative yield. In fact, Holt and Birch (1984), describing plant resistance to three aphid species in relation to the taxonomy of the genus Vicia and the degree of domestication of the host, concluded that within each taxonomic group, the most domesticated species offered the best conditions for aphid growth. Plant selection based on high yield did not always preserve the genes responsible for plant defence mechanisms (Clarke, 1984). This is why the use of pesticide in plant protection became dominant in the years immediately following the World War II (1939-1945), during which time the chemical industry was at its peak.

The damage of pesticides to the environment (OMS, 1991; Ramade, 1982; Zweig and Aspelin, 1985; Weir, 1986), the steady rise in the costs of new pesticide development, coupled with an exponential increase in the number of insecticide-resistant insect species led to increasing awareness of the value of biological control schemes. In entomology, the development of resistance to many insecticides (Painter, 1958) saw thoughtful entomologists conclude that the search for sources of insect resistance in reducing the populations of insects and the damage caused by them belong in all well-rounded insect control projects.

Focus on plant health is gaining on the traditional approach of a systematic attack on, and destruction of, diseases and pests. Rather than attempting to achieve one hundred percent protection from pathogen or pest attack, the challenge is discovering the means of securing the plant's health, for example by mobilizing the plant's own defence system against biotic and abiotic stress factors, and thereby, guaranteeing quantitative as well as qualitative yields. Consequently, the search for innovative methods has become a key issue in plant protection sciences.

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The notion that plants may be able to develop acquired immunity to infection, following exposure to a pathogen or antigenic substances derived from a pathogen, has existed ever since discovery of the animal immune system in the latter years of the nineteenth century, when it was proven that plant vaccination could lead to changes in the response of tissues to subsequent microbial challenge (Lucas, 1999). Studies of acquired immunity in plants were reported by Ray (1901) and Beauverie (1901). Following the literature review by Chester (1933) and Gaeumann (1951) on the activation of plant defence mechanisms, many publications began to discuss the specific effects of different types of induced resistance.

Induced resistance was subsequently the focus of many experiments. It was obtained in cucumber against Colletotrichum lagenarium (Kuc et al., 1975), in tobacco leaves against Pseudomonas solanacearum (Squeira and Hill, 1974), as well as against TMV and other viruses in tobacco (Ross, 1961; Loebenstein, 1972). Giebel (1982) intensively researched the mechanisms of induced resistance against nematodes, while Karban (1986) demonstrated that induced resistance against spider mites (Tetranychus spp.) was possible in cotton seedlings that either had been mechanically abraded or had primary infections. Agrawal (1998) observed that experimentally induced plants consistently received less herbivory than untreated ones. The responses of induced resistance in plants, discussed in the literature can be summarized in three main points:

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Many studies have been carried out to explain the mechanisms of resistance in plants following pathogen or pest attack. The mechanisms are characterized by de novo synthesis of proteins and secondary metabolites, or by the accumulation of already synthesized compounds, together referred to as induced responses (Karban and Baldwin, 1997). Monocotyledon and dicotyledon plants respond to fungal attack with a complex network of defence mechanisms (Dixon and Harrison, 1990), which include the synthesis of polymers, forming physical barriers (cutin, lignin, and callose), antimicrobial metabolites (phytoalexins), and pathogenesis-related proteins (PRs) (Jach et al., 1995).

Pathogenesis-related proteins were initially defined as proteins encoded by the host plant that were induced only in pathological or related situations (Collinge, 2001). Today they have a wider definition, and include proteins induced by wounding and pest attack (van Loon et al., 1994). Induction of these proteins occur both locally and systemically upon pathogen infection, herbivore-attack wounding or under the influence of other stress factors. The biochemical and molecular characteristics of the PRs are classified into 14 families (van Loon and van Strien, 1999) where PR-6 proteins are defined as proteinase inhibitors (PIs) involved in the defence against insects and other herbivores, micro-organisms, and nematodes (Koiwa et al., 1997; Ryan, 1990).

PIs are the most extensively studied induced defence proteins (Ryan, 1990, 1992) and there is increasing evidence supporting defensive roles for proteinase inhibitors against insects (Ryan, 1990). The applicability of this paradigm to piercing-sucking insects has remained undetermined (Jongsma and Bolter, 1997), because these insects appear to lack endoproteinase activity within their tract (Rahbé et al., 1995) and therefore have not been considered likely targets for PIs. However, Casaretto and Corcuera (1998) noted that after infestation of barley by Schizaphis graminum and Rhopalosiphum padi the accumulation of chymotrypsin inhibitors was two-fold higher in resistant plants compared to susceptible ones. Adding plant PIs to artificial diet, affects the survival of Diuraphis noxia, S. graminum and R. padi (Tran et al., 1997). Other types of PR proteins intensively studied for their involvement in resistance response include chitinase, β-1,3-glucanase (Dassi et al., 1996) and peroxidase, (Esnault and Chibar, 1997; Gaspar et al., 1982; Moerschabacher, 1992).

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Chitinase is cited as the main proteinaceous inhibitor of fungal growth in bean leaves and can be induced by the plant hormone ethylene, or by pathogen attack (Schlumbaum et al., 1986). The activity of chitinase against pests has also been demonstrated; lectin of Brassica spinescens and the closely related agglutinin from wheat germ and nettle show significant insecticidal activity when given to Brevicoryne brassicae in chemically defined synthetic diets (Cole, 1994). Since chitinase and β-1,3-glucanase are capable of degrading fungal cell walls in vitro (Mauch et al., 1988), these enzymes have received particular examination as possible defence compounds in plants against fungi containing glucan and chitin in their cell walls (Lusso and Kuc, 1996).

In plant defense reaction to pathogens, however, the precise role of peroxidases is not well understood. In vitro essays indicate that their presence with a suitable hydrogen donor and H2O2 can produce toxic products (e.g. oxidized phenols)

that are lethal to infecting microbes (Esnault and Chibbar, 1997). Dowd and Lagrimini (1997) reported that a caterpillar species absorbed much less from Nicotiana spp. overproducing tobacco anionic peroxidases than the wild type. Additionally, van der Westhuisen et al. (1997), investigating the role of intercellular peroxidase and the chitinase activities of three wheat cultivars [Tritium aestivum L. cvs “Tugela DN”, “Molopo DN” and “Betta DN”], concluded that these compounds possibly play a role in resistance against the Russian wheat aphid (D. noxia).

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There is strong evidence that the activities of β –1,3 -glucanase (Ji and Kuc, 1995), chitinase (Irving and Kuc, 1990; Metraux and Boller, 1986; Metraux et al., 1989) and other types of PR proteins correlate with induced resistance. However, Forslund (2000), who predicted β -1,3 -glucanase and chitinase as induced resistance factors in barley Hordeum vulgare to Rhopalosiphum padi, could not establish such a correlation.

Many other compounds are cited in the literature as induced resistance factors against aphids. In cereals, hydroxamic acids (Niemeyer, 1988), gramine (Kanehisa et al., 1990; Zugina et al., 1988), phenols (Leszczynski et al., 1989) and aconitic acid (Rustamani et al., 1992) have been cited as compounds involved in resistance against different aphid species and could be investigated as natural chemicals that offer possibilities for the control of other insects and pathogens (Corcuera, 1990).

A body of research supports the idea that, in local and systemic induced resistance processes, the mediation of the inducers is necessary for the signal transduction and the expression of specific genes in the induced defence response of the plant. The inducers cited in the literature can be categorized into those of biotic and those of abiotic origins. Biotic inducers are classified into pathogen, apathogen or endophyte type, while abiotic inducers are composed of pure substances or substances of complex nature. Thus, of abiotic inducers, jasmonic acid (JA) and salicylic acid (SA) are involved in the signalling pathways that allow plants to express induced resistance against pathogens and pests. Jasmonic acid has been shown to have an effect in the signal transduction cascade, mediating inducible plant defence responses against herbivore attack (Enyedi et al., 1992; Sembdner and Parthier, 1993; Mueller et al., 1993). Further, application of JA and its volatile methyl ester jasmonate (MeJA) to plants triggers the synthesis of a number of defence-related secondary metabolites in plant: PIs (Farmer and Ryan, 1990; Koiwa et al., 1997), polyphenol oxidase (POX) (Schaller and Ryan, 1995), thionins (Epple et al., 1997), osmotin (Xu et al., 1994) and alkaloids (Baldwin, 1997; Zhang and Baldwin, 1997). In signal transduction for systemic acquired resistance (SAR), salicylic acid (SA) has been suggested as the signal chemical because its concentration rises dramatically after a pathogen infection (Malamy et al., 1990; Métraux et al., 1990). Raskin (1992) and Hildebrand et al. (1993) have reported that topical application of salicylic acid increases plant resistance to herbivores.

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Besides the synthetic inductors, jasmonic acid, salicylic acid (Conti and al. 1996) and the 2,6-dichloroisonicotinic acid (INA) (Metraux et al. 1991; Thieron et al. 1995) or trigonellin (N-Methyl nicotinic acid) (Kraska and Schönbeck, 1993), a variety of other natural inducers are often cited in the literature. Hence avirulent pathogens (Chaudhary et al. 1983, Villich-Meller and Weltzien, 1990; Olivain et al. 1995), plant extracts (Herger et al., 1988), mycorrhiza fungi (Schönbeck and Dehne, 1979; Bargmann and Schönbeck, 1992), rhizobacteria (Defago et al., 1995; van Loon, 1995), and metabolites from bacteria (Maiss, 1987; Steiner, 1990; Steiner and Schönbeck, 1995; Leeman et al., 1995) can serve as induced resistance agents in plants.

Bacillus subtilis, referred to as a Plant Growth Promoting Rhizobacterium (PGPR), has been the subject of investigation in numerous laboratories, because its application is often associated with an increase in plant growth rate. PGPR application to crops or soil for beneficial effects was first reported in 1950s in studies conducted in the former Soviet Union, and later in the West (Zehnder et al., 2001). It was initially thought to enhance crop fertility by increasing the amount of available nitrogen (Cooper, 1959). Additionally, Idris et al. (2002) recently demonstrated that some strains of B. subtilis are able to degrade phytate (myo-inositol hexakisphosphate), the major storage compound of plant phosphorus, thus liberating phosphorus for improved plant growth. Besides its PGPR qualities, B. subtilis has been reported as having a wider application as a biological control agent, capable of suppressing soilborne pathogens (Dunleavy, 1955; Broadbent et al., 1971; Schippers et al., 1987; Bochow, 1992; Kloepper, 1993; Bochow et al., 1995; Doley S., 1998). Consequently, the role of Bacillus subtilis as induced resistance or tolerance agent is receiving considerable investigation.

While the mechanisms that sustain such activity are not well understood, some suggestions have been made. Scheffer (1983) and Voisard et al. (1989) have argued that PGPR strains may activate host defense systems, based on a lack of direct antibiosis of the strains against the pathogens, or on the correlation of biocontrol with plant growth promotion. Direct evidence supporting the hypothesis that PGPR strains, which remain on plant roots, can induce resistance in plants to foliar or systemic pathogens was found in three pathosystems: cucumber and anthracnose (Wei et al., 1991), carnation and Fusarium wilt (van Peer et al., 1991), and bean and halo blight (Alstroem, 1991). The mechanisms by which PGPR strains exhibit biological control over soil pathogens include antibiosis through bacterial production of antifungal compounds, competition for ferric iron, competition for infection sites, and production of lytic enzymes (Kloepper, 1993). Some proteins and protein complexes, as well as protease and ammonium play a key role in induced resistance processes in the plant (Bochow, 1998).

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The use of culture filtrate of Bacillus subtilis as induced resistance agent is gaining interest in many laboratories. It was used to induce resistance against virus in cucumber and barley (Maiss, 1987). Steiner and Schönbeck (1995) discussed the capacity of Bacillus subtilis' culture filtrate to induce resistance in mono cotyledons against plant biotrophic pathogens. Raupach and Kloepper (1996) noted a reduction in cucumber mosaic virus (CMV) infection intensity in cucumber plants following Bacillus subtilis application. After treatment with metabolites of Bacillus subtilis strain B50, barley was found to be resistant against Erysiphe graminis (Schönbeck et al., 1982; Kehlenbeck et al., 1994). It also induced resistance in wheat against Sitobion avenae but not against Rhopalosiphum padi (Galler et al., 1998).

In this research, three strains of B. subtilis, FZB24, FZB37 and FZB38, produced by FZB Biotechnik GmbH, Berlin, were used. While the strains FZB37 and FZB38 are still under investigation, the strain FZB24, tested many times in both in vivo and in vitro tests, has been widely reported as a plant pathogen inhibitor and plant growth stimulator bacterium (Schmiedeknecht, 1993; Schmiedeknecht et al., 1994, 1995, 1996; Bochow, 1995; Fey, 1996; Grosch and Junge, 1996).

Investigation of the role of Bacillus subtilis and its metabolites as induced resistance agent against pests has barely begun. While the mechanism by which induced resistance operates is not yet well understood in the majority of host-parasite systems, some researchers, such as Bochow (1995) and Schönbeck et al. (1993) have formulated the mode of action of induced resistance, which can be summarized in the following steps:

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The absence of direct effect of the inducer on the target pathogen or insect characterised the fundamental difference between the inducer and conventional pesticides. While classical pesticides are known to act directly on the target organisms, inducers are understood to promote or enhance the host plant’s already existing defense system to resist or tolerate any biotic or abiotic stress factor. The various inducer agents identified by the researchers and referred to in the literature are all reported to be active via the plant's own defense mechanisms. Exogenous application of inducers is presumed to not have a direct effect on the target pathogens or insects. The acid, 2,6 dichloroisonicotinic, has no direct effect on bacterial or fungal pathogens, but induces the same set of defense genes that are systemically activated by local pathogen infection (Ward et al., 1991).

Given the biochemical characteristics of plant secondary metabolites and the fact that induced resistance relies upon the activation of these, there is a variable interval of time between the application of the inducer agent and the plant biochemical response. In ethylene-treated plants, chitinase activity began to increase after a lag of 6 hours and induced 30-fold within 24 hours (Boller et al., 1983). Induced resistance response in cucumber leaves against Colletotrichum lagenarium required 24 hours (Dean and Kuc, 1985). Induction of PR proteins was recorded as early as 25 hours after inoculation and their quantity increased for up to 72 hours following the application of salycylic acid in the form of spray onto the surface of barley leaves (Tamas and Huttova, 1996).

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The unspecific character of the above inducer presents an opportunity to obtain induced resistance in different plant species against a range of pest systems. Nevertheless, this quality of the inducer is not valid in every case and researchers have made contradictory observations. For instance, wheat plants treated with B. subtilis strain B50 demonstrated resistance to Sitobion avenae but not to R. padi (Galler et al., 1998).

The systemic character of the inducer is defined as the capacity of the inducer to transduce its effect from the treated part of the plant to the untreated one and thereby ensure the whole plant is protected. Ross (1961) showed that inoculation of one leaf of a tobacco cultivar possessing local lesion resistance to tomato mosaic virus (TMV) increased the resistance of other leaves on the same plant to TMV, while Staub et al. (1992) reported that tobacco plants treated with two isonicotinic acid derivatives at 200 ppm, by injecting the two lower leaves, were protected systemically against tobacco mosaic virus (TMV), Pseudomonas syringae pv. syringae, Tabaci (Wildfire), and other plant viruses. Induced resistance can be also obtained via local induction where only the inoculated plant parts are protected against subsequent infection. Some compounds known to induce resistance are quoted as acting only locally, not systemically. Ryals et al. (1996) observed that exogenous application of SA will induce resistance only in leaves that are treated with the compound. In both cases, systemic resistance and locally induced resistance, the dose of the inducer agent is reported to not be stochiometric in relation to the plant defense response. Schönbeck et al. (1993) observed that, while the concentration of trigonelline had increased, this did not lead to an increasing efficiency of induced resistance against powdery mildew (Erysiphe graminis f. sp. hordei) in barley.

Induced resistance mechanisms in host plants against parasites rely on a complex process in which the latent resistance genes of the plant are activated and coupled with reinforcement of the already expressed resistance genes (Schönbeck et al., 1993).

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The promotion of plant growth as a result of treatment with Bacillus subtilis and its metabolites, and the following change of phytohormonal balance, are understood to play a key role in the preservation of plant health (Bochow et al., 1995; Doley and Bochow, 1996).

The objective of this thesis was to assess whether topically treating Triticum aestivum and Vicia faba, respectively mono and dicotyledonous plants, with Bacillus subtilis and its metabolites led to induced resistance against Rhopalosiphum padi and Aphis fabae.

To elucidate the mechanisms of possible induced resistance resulting from the treatments, two physiological tests were conducted. Firstly, the chlorophyll fluorescence of plants pre-treated with B. subtilis and its metabolites was measured to assess how the inducers may compensate for the pathological sink created by the feeding activity of aphids. Secondly, qualitative and quantitative assessments of the amino acids were performed.

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The methods used to investigate the objectives established in this research were chosen for their reliability in providing clear answers to the complexity of the interaction between the test insects and their host plants.

Bacillus subtilis metabolites were assayed against Uromyces appendiculatus on Vicia faba to ascertain to what extent they can inhibit the development of these biotrophic fungi. In view of the fact that parallels are often drawn between the mechanism of aphid- plant and pathogen- plant attack (Dreyer and Campbell, 1987), it was strongly hypothesised that the same metabolites could be used in induced resistance tests to aphids in this study.

To assess the performance of individual aphid feeding on pre-treated plants with B. subtilis and its metabolites, a life table model test was designed to measure insect growth parameters. A widely used method in assessing plant resistance to aphids (Wojciechowicz-Zytko and van Emdem, 1995) applies the measurement of individual weight gain over the time, the Relative Growth Rate (RGR) (Fischer, 1921; van Emden, 1969) and the intrinsic rate of natural increase (rm) (Wyatt and White, 1977). Correlation between RGR and rm values has been completed for some 12-aphid species, including R. padi (Kempton et al., 1980; Leather and Dixon, 1984) and Aphis fabae (Dixon, 1990; Guldemond et al., 1998).

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Our study assumed that the pre-treated host plants (Vicia faba and Triticum aestivum) would exhibit resistance characterized either by nonpreference (antixenosis), where the host plant deters aphids from settling and colonising them, or antibiosis, where the increase rate of aphids is restricted (Painter, 1951; Kogan and Ortman, 1978; Dixon, 1987). Host plant resistance is a major strategy in aphids control - if plant resistance increases, this can extend the duration of aphid development, as well as slow down the aphids' reproduction rate (Dreyer and Campbell, 1987). After plants had been exogenously pre-treated with the metabolites of B. subtilis and we had observed that this effectively resulted in resistance against aphids, we attempted to elucidate its mechanisms. Acute toxicity and artificial diet tests for the R. padi and A. fabae were conducted, in order to localize the effect obtained in the induced resistance treatment and to establish whether this effect was a result of a direct contact between the tested aphids and the metabolites, or whether it was mediated via the plant. The results led us to conclude that the acute toxicity and artificial diet tests did not appear to influence the growth parameters of the tested insects and that the induced resistance was most probably due to physiological change following the treatment with B. subtilis metabolites.

Two types of physiological tests were then carried out:

Aphid feeding has been reported to induce a pathological sink in their host plant in concurrence with the natural sink. The term “sink” is defined by Lafitte (1984), as the regions of the plant that import photosynthate, whereas 'sources' are organs that export photosynthate. Fouché et al. (1984) and Al-Mousawi et al. (1983) observed cereal crop damage occurrence during feeding when the aphids inject phytotoxic substances into leaves and remove assimilates from leaf vascular tissues.

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The first objective was to quantify the changes in the chlorophyll fluorescence caused by aphid feeding on young seedlings of Vicia faba and Triticum aestivum plants that had been induced by B. subtilis and its metabolites, as well as assess the resistance of those induced plants compared to the control (water-treated plants) by means of aphid biomass analysis.

Secondly, the free amino acids of the Vicia faba seedlings were investigated. Thiswas prompted by the frequent citing of primary and secondary compounds as playing important roles in resistance factors to aphids. Primary compounds, e.g. amino acids, may in some part explain the resistance of some oat and barley varieties to Rhopalosiphum padi (Tsumuki et al., 1987; Weibull, 1988). Van Emden (1972) could establish, for Myzus persicae and Brevicoryne brassicae, that amino acids and the allylisothicyanate of the plant analysed by him accounted for a large proportion of observed variability in the mean performance of these aphids. The development of chemically defined diets for aphids (Mittler and Dadd, 1962; Auclair and Cartier, 1963; Griffiths et al., 1975) has made it possible to vary the concentration of single compounds in aphid food in order to test the effect of this variation on aphid growth rate and fecundity. The studies of V. faba cultivars by Poehling and Morvan (1984) have indicated that free amino acids are partial determinants of resistance. Weibull (1987) also observed that the relative growth rates of R. padi closely followed the phenological change of free amino acids in phloem sap of oat and barley plants as they aged. Srivastava (1987) has proposed three categories of amino acids as important in aphid nutrition. The first category functions as a phagostimulant, the second may possibly have slight inhibitory effect, while the third category has a strong inhibitory effect. When the third category is dominant in the sap, this may decrease feeding and contribute towards the resistance of the plant. Much relevant literature argues that the quantitative concentration of amino acids has less effect than its qualitative variation. Generally, 10 amino acids are considered to be essential in insects’ nutrition (Dadd, 1985). Aphids are vicious pest insects; it has even been demonstrated that they manipulate their host plant's nutritional quality by changing amino acid levels (Sandström et al., 2000). Therefore, the phloem amino acids of V. faba plants were measured, in order to pinpoint the extent of influence of B. subtilis metabolites on the pre-treated aphid’s host plant.


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