The here presented results show that in all the studies, induced resistance was possible in both host plants, summer wheat (Triticum aestivum) and broad bean (Vicia faba), where the supernatant of B. subtilis was used. This effect was not caused by direct toxicity of the supernatant to the insects. This was demonstrated in the acute toxicity test, where aphids were directly exposed to the supernatants. The culture filtrates of B. subtilis were used in the artificial diets to observe whether the relative growth rate of A. fabae would be affected. Obviously it was not possible to introduce the supernatant into the artificial feeding test since the diet was maintained as sterile. While the chlorophyll fluorescence measurement in the treated plant leaf experiments showed better trends, it was not significantly affected. Conversely, introducing the spore suspensions via seed treatment into sterile soil, T. aestivum seedlings showed significant higher chlorophyll fluorescence compared to the control (water treatment). In order to understand all the observations noted here and the established induced resistance against A. fabae and R. padi, the discussion will focus on the following points:
Vicia faba - Uromyces appendiculatus
Before testing B. subtilis and its metabolites against aphids, it was important to establish the effect of our strains against a biotrophic pathogen. The use of Uromyces appendiculatus here confirmed that the culture filtrate and supernatant of B. subtilis strains FZB24, FZB37, and FZB38 can reduce the development of urediospores. Other researchers have already reported that B. subtilis and its metabolites play such a role. The culture supernatant and other metabolites of B. subtilis have been found to have a higher inhibitory effect on the number of Uromyces phaseoli pustules of more than 90% (Baker et al., 1983; Mizubuti et al., 1995), as well as on Uromyces appendiculatus (Baker et al., 1985; Bettiol et al., 1992). Although the mode of action is not completely understood it is suggested that an inhibitory metabolite or a toxic substance that suppressed conidial germination is involved. Since our experiment showed that systemic treatment with culture filtrate and supernatants of B. subtilis strains FZB24, FZB37, and FZB38 is effective in reducing urediospores of Uromyces appendiculatus, one can assuredly state that the inhibitory effect is mediated via the plant system itself. Any toxic effect due to direct contact between the urediospores themselves and the bacteria metabolites can be excluded. The effect can be explained by a physiological change in the plant sap that prevents Uromyces appendiculatus from developing normally. As we did not further investigate the causes of this inhibitory effect, we have to rely on existing research, which stipulates that, despite the differences in food uptake between Uromyces appendiculatus, a biotrophic fungus, and aphids, that is, phloem sucking insects, both require the same form of nutrient, based on the mobilisation of amino acids on their host plants.
Vicia faba – Aphis fabae and Triticum aestivum - Rhopalosiphum padi
Aphids are economically significant pests of arable plants. Of approximately 4,000 species and closely related genera in the world, about 250 are serious pests. Aphids feed by sucking plant sap which results in direct plant damage by reducing the plant's resources (Dreyer and Campbell, 1987); they can cause disease in the plant by disrupting the tissues, injecting toxins, and by transmitting viral diseases (Dixon, 1987; 1978; Pickett et al., 1992). It is estimated that 60 % of all plant viruses are spread by aphids (Schwarz, 1985). Unlike many other insects, aphids can reproduce without mating (parthenogenesis) and, given their high reproductive rates and short maturation times, explosive increases in aphid populations can occur in very short periods (Dixon, 1987; 1978). The above-mentioned characteristics demonstrate that the aphid is a serious pest and a limiting factor in crop production. Today most aphid control methods depend heavily upon the use of chemicals and, despite early success stories, numerous aphid species have developed resistance to aphicides (Devonshire, 1989). Thus looking for a new approach to aphid control is of great interest to agriculture.
The experiments discussed here were able to establish induced resistance to A. fabae and R. padi in greenhouse conditions. In both studied insects, A. fabae and R. padi, the characteristics of the resistance were similar. Aphids feeding on supernatant of B. subtilis strains FZB24, FZB37 and FZB38 induced plants had longer development times (tD) and pre-reproduction (td) times, with accordingly lower relative growth rates (RGR) and intrinsic rates of natural increase (rm). This kind of resistance to aphids, when growth and development of the insect are negatively affected, is usually termed as antibiosis. It has been reported by other researchers as well as utilized in aphid population dynamic study. Birch and Holt (1984), studying the sources of resistance to aphids in Faba bean and its wild relatives, found that longer aphid development times and lower reproductive rates were the key factors involved in antibiosis. The maximum possible rate of population increase that occurs when there are no biotic restraints on the aphids is the intrinsic rate of natural increase (Birch, 1948). Large individuals of A. fabae are reported to be more fecund and have a higher reproductive rate than small individuals (Dixon and Wratten, 1971; Taylor, 1975; Dixon and Dharma, 1980a) and therefore those factors that affect growth and development and thus govern adult size also primarily determine reproductive rate and fecundity (Dixon and Dharma, 1980b).
Our research found that the supernatants of B. subtilis strain FZB24, FZB37 and FZB38 limit the increase of A. fabae and R. padi, whose performance was affected when feeding on pre-treated host plants.
The description of antibiosis correlates with our observations of the performance of A. fabae and R. padi following the pre-treatment of their host plants with supernatants of B. subtilis strains FZB24, FZB37 and FZB38. As defined by Painter (1951), antibiosis takes place when a plant becomes resistant by exerting an adverse influence on the growth and survival of the insect. These effects are characterized by the reduced survival of the insects or reduced weight and size with longer time taken to complete the lifecycle, as well as a reduced growth rate and reduced fecundity. This type of resistance is different from antixenosis or nonpreference in which a host plant displays a degree of resistance by exerting an adverse effect on the insect’s behavior (Beck, 1965).
Beck (1965) points out that a distinction must be drawn between resistance to feeding and resistance that acts by interfering with the physiological processes underlying growth, metamorphosis, and reproduction, since such physiological effects may be caused by metabolic inhibitors in the plant tissues, or by the plant’s failure to provide either specific nutrients or the nutrient balance required by the insect. Additionally, these two types of resistance, "nonpreference” and “antibiosis” can coexist since they are above all empirical values. We cannot easily distinguish between these two values, as we did not investigate the feeding behavior of the aphids with the use of electronic monitoring test methods. Investigating the role of hydroxamic acids in resistance to R. padi on cereal, Givovich et al. (1992) reported that a high mesophyll concentration of hydroxamic acids may provide a level of feeding deterrence during stylet penetration, while a phloem sap concentration may also provide deterrence combined with antibiosis. The causes of resistance to aphids can be multiple and each aspect of the aphid's relation to its host plant when feeding on plants with induced resistance is of interest. Electronic monitoring of aphid feeding has been mostly used to investigate host plant resistance or suitability (Adams et al., 1982; Haniotakis et al., 1978; Kennedy et al., 1978; Montllor et al., 1990; Shanks and Chase, 1976). However, as quoted by Montllor (1991), behavior does not necessarily differ between good and poor hosts, though some correlation between behavioural characteristics and performance parameters might be expected.
While the supernatants of B. subtilis strains FZB24, FZB37, and FZB38 were able to induce resistance against A. fabae and R. padi (Tables 11, 12, 13, 14), the use of other metabolites as culture filtrate, vegetative cells and spore suspensions of the same bacteria and strains presented different results.
Indeed, induced resistance after foliar treatment with culture filtrate, spore suspensions and vegetative cells of B. subtilis was not possible (Tables 5, 6, 7, 8, 9, 10, 15, 16, 17, 18). On the basis of the life table of A. fabae and R. padi, the growth parameters seem to be negatively affected in some cases; however this was not statistically confirmed. Available research has confirmed the inhibitive effect on plant pathogens from B. subtilis culture filtrate. Doley (1998) reported an increased of β-1, 3 glucanase activity in tomato seedlings treated with culture filtrate at the logarithmic and transition phases of B. subtilis strain (FZB24). While β-1, 3 glucanase was cited in induced resistance cases, this was not verified in our research. The introduction of culture filtrate in the artificial diet also did not show any influence on the relative growth rate of A. fabae. The results observed here show that there is a qualitative difference between the supernatant and the culture filtrate of B. subtilis.
B50 culture filtrate of another strain of B. subtilis also could not induce resistance to A. fabae in our experiment (Tables 5, 6). This culture filtrate was, however, found to induce resistance in wheat plants against Sitobion avenae but not against R. padi (Galler et al., 1998). An explanation could be the qualitative differences between the culture filtrates of these two different strains of B. subtilis. It has been demonstrated that the antagonistic qualities of B. subtilis are strain dependant (Schönbeck et al., 1971; Broadbent et al., 1971; Swinburne et al., 1975; Krezel and Leszczynska, 1978; Utkhede, 1984; Tschen and Kuo, 1985; Huber et al., 1987; Hiraoka et al., 1992; Orihara et al., 1995; Asaka and Shoda, 1996).
Aphids as sap suckers are known to be highly sensitive to changes in the plant hormonal balance. Chatters and Schlehuber (1951) observed that the cell walls of the phloem cells and their contents were stained differently in those on which aphids were feeding, than in control tissues, which suggests that changes occur in the chemical composition of infested tissue. Aphids' distribution between and on the host plants is largely determined by variations in the quality of phloem (Dixon, 1985), and aphids that reach the phloem of resistant cultivars of their host plant tend to cease feeding shortly after the phloem is penetrated (Nielson and Don, 1974). While it is mentioned that some aphid species may prefer feeding on mesophyll tissue (Lowe, 1967), the primary food of aphids remains the phloem sap (Montllor, 1991). The process of host selection by aphids is characterized by a series of steps, involving a range of cues. Klingauf (1987) divided this process of host selection into the following: attraction, testing of the plant surface, penetration and testing the phloem.
The phloem long-distance translocation system of plants appears to function both as a nutrient delivery system and as an information superhighway (Zimmermann, 1960; Eschrich and Heyser, 1975). The central role of the phloem in the translocation of nutrients has long been recognized. Aphids feeding on its host plant are understood to secrete substances into plants that rapidly induce changes in growth and translocation. Many of these changes in plant metabolism are to the aphid’s advantage (Dixon, 1975). Wittmann (1995) observed that, following R. padi infestation, the saccharose and glutamic acid content of wheat seedling leaves were found to be reduced. He concluded that the reduction of amino acids concentration in infested wheat seedlings was mainly due to the feeding habits of R. padi, in which a pathological sink was established in its host plant. Other researchers have come to the same conclusion on the question of aphid feeding. Thus Hawkins et al. (1987) pointed out that Acyrthosiphon pisum and Aphis craccivora damage to legume plants can independently induce a new sink-source relation to their advantage. We believe the role of the supernatants of B. subtilis strains (FZB24, FZB37 and FZB38), used in research as induced resistance, has been to react against such manipulation and preserve the plant integrity. Correspondingly, it was possible to show that A. fabae and R. padi could not use V. fabae and T. aestivum respectively to their advantage since the growth parameters of these aphids were largely influenced by the treatments with supernatants of B. subtilis. Even though behavioural studies were not conducted in this research to evaluate further resistance mechanisms of supernatant-induced plants to aphids, we would presume that such tests would demonstrate variation between treated and untreated plants. An electronic monitoring technique used by Dreyer et al. (1987) to measure aphid probing showed that initially, the length of probing time required to reach the phloem on an aphid-resistant line is longer than as on a corresponding susceptible line. Galler (2001) made the same observation, reporting that S. avenae feeding on B50 induced plants needed a longer period of time to reach the phloem compared to S. avenae from non-induced plants.
Following foliar treatment with supernatant of B. subtilis FZB37, the chlorophyll fluorescence in both plant systems demonstrated a better yield (Tables 24, 26). The fresh weight and dry weight of aphids were reduced; this, however, was found to be statistically significant for A. fabae but not for R. padi (Tables 25, 27). One explanation could be the small number of replications used in the experiments.
The seed pre-treatment with spore suspensions of B. subtilis strain FZB37 showed a positive trend with a better chlorophyll-fluorescence level measured in the plant leaves and a diminished fresh weight and dry weight of A. fabae and R. padi feeding on respective host plants (Tables 28, 29, 30, 31).
Total concentration of free amino acids in phloem sap
The increase in the total concentration of free amino acids in supernatant and culture filtrate treated plants was not statistically verified (Figures 23, 24) pre- and post-infestation with A. fabae. The higher level of amino acids found in V. faba seedlings (Figure 23) following the supernatant treatment could be attributed to the growth-stimulating qualities of the inducers used in this research on the plants. It has frequently been observed that the total concentration of free amino acids in the plant does not necessarily correlate with resistance to aphids. Sandström and Pettersson (1994), studying the resistance of Acyrthosiphon pisum on pea genotypes, concluded that the total concentration of amino acids in the phloem sap was not responsible for the differences in the performance of the aphid. The higher level of total concentration of free amino acids in the treated plants cannot influence the change in individual amino acids since this variation was noticeably significant once the A. fabae started to feed. The individual variation of amino acids here observed could be explained by the feeding behaviour of A. fabae, which acts as a sink in mobilising the nutrient from plant source organ (Figures 25, 26).
Concentration of individual amino acids in phloem sap
Several researchers have studied the amino acids required for general insect and particularly aphid growth. Many different amino acids, including arginine, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, thrytophan and valine, have been referred to as essential for insects (Dadd, 1985). Acyrtosiphon pisum requires the presence of arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine for growth (Retnakaran and Beck, 1968) and Turner showed that Aphis gossypii required sulphur containing amino acids and cysteine. Dadd and Krieger (1968) in their basal diet for Myzus persicae mentioned the doubtlessly important role of serine for growth. They substituted eight of the amino acids included in standard diet with large amounts of serine or alanine and concluded that, in the case of alanine, the lowest level provided the best growth, whereas with serine, double concentration was optimal and enabled aphids to achieve weights only slightly inferior to those achieved on the standard diet. This obviously speaks for the importance of serine in aphids' nutrition. Essentially eight amino acids are considered as important for the development of A. fabae when reared on synthetic diets (Leckstein and Llewellyn, 1973). Thus the two authors found that after the individual omission of alanine, histidine, methionine, proline or serine, A. fabae diet intake was lower than that of the complete diet. The omission of cysteine, phenylalaline, or tyrosine failed to reduce diet intake. Other researchers have categorized the amino acids involved in aphids feeding. Weibull (1987) observed that the variation in amino acids composition alone was sufficient to create separate levels of resistance, expressed in as differences in population growth between cultivars.
In A. fabae treatments with supernatant and culture filtrate of B. subtilis strain FZB37 the concentration of individual phloem amino acids of V. faba changed, as indicated in Figure 25. Among the essential amino acids for A. fabae to which we have referred, the serine concentration in supernatant treatment, which does not vary when comparing Figures 25 and 26, was found to be significantly lower when compared to the control (water treatment) and the culture filtrate of B. subtilis strain FZB37.
The fact that the concentration of serine did not change in supernatant FZB37 treated plants could suggest that the integrity of this amino acid was preserved in the phloem sap following the treatment. As reported by Ryan (1990) most animals require proteolysis to degrade and use the component amino acids of the proteins they consume. The unchanged concentration of serine in FZB37 sup induced plants in our study could be interpreted as a failure of the serine protease produced by A. fabae to degrade the plant protein and liberate enough serine for the aphids' growth, development and reproduction. Wolfson and Murdock (1990) discuss the importance of serine in insect nutrition, finding that insects mainly use one or a combination of serine, cysteine and aspartic acid as major digestive proteolytic enzymes. Furthermore, it is said that inhibitors of these enzymes are produced by plants, and presumably modulate the growth and development of pests by attenuating protein degradation (Koiwa et al., 1997). At the same time aphids are said to be able to replace the missing amino acids in its diet supply, probably via the intracellular symbionts (Srivastava et al., 1984). Indeed, omission of the usual ten essential amino acids from the diets of larvae of M. persicae, derived from mothers treated with antibiotics, resulted in a considerable reduction in growth (Mittler, 1971). It is even suggested that aphid symbionts may synthesize the missing amino acids. While our research did not quantify to what extent serine was supplied to the A. fabae, it did demonstrate that this aphid did not overcome the negative effects linked to the host plant pre-treated with supernatant of B. subtilis strain FZB37.
Proteinase inhibitors (PIs) have often been associated with resistance against pathogens (Peng and Black, 1976; Roby et al., 1987) and herbivores (Urwin et al., 1995; Johnson et al., 1989) in relevant literature. In experiments attempting to identify new resistance compounds against aphids, PIs are cited as potential and interesting materials. Five plant proteinases were tested for the ability to control Diuraphis noxia, Schizaphis graminum, and R. padi. Thus it was found that though the effectiveness of the inhibitors varied, the proteinase inhibitors from potato were potentially effective proteins for the control of these species (Tran et al., 1997). More specifically, serine proteinase inhibitors were cited as the main factor in inducible resistance of tomato foliage to lepidopteran larvae (Johnson et al., 1995; Stout et al., 1996). Proteinase inhibitors were also said to be detrimental to the growth and development of insects from a variety of genera including Heliothis, Spodoptera, Diabiotica and Tribolium. A body of research has cited serine proteinase inhibitors as one of the inducible defensive mechanisms that plants have evolved to inhibit the digestive enzymes of piercing-sucking insects and microorganisms (Green and Ryan, 1972; Ryan, 1978; Hilder et al., 1987; Johnson et al., 1989).
Although our research did not investigate the phloem sap for PI proteins, it is tempting to speculate that serine proteinase inhibitors could be found in supernatant FZB37 induced plants as the concentration of serine has remained the same in supernatant FZB37 induced plants as shown in Figure 24.
In the interaction plant-insect, two main strategies seem to be involved. On one hand, the plant has to evolve mechanisms to protect the operational integrity of the phloem, which contains the flow of nutrients and information necessary for further growth and development, and on the other, the insect looks for the same nutrient through proteolysis, using enzyme proteases, trying to mobilise amino acids for its growth, development, and reproduction. In this competition for the nutrient, the inducer functions as a group of signalling molecules, capable of activating the expression of genes, which can help the plant to maintain or even boost its defense integrity. Piercing-sucking insects, whose primary nutrition is gained through uncontrolled access to the phloem sap, pose a dangerous challenge to the integrity of the phloem system. It is interesting to note that numerous plant PIs have been demonstrated to modify plant-arthropod interactions, via their role as digestibility reducers, toxins, or modifiers of feeding behavior (Broadway et al., 1986; Duffey and Stout, 1996). The applicability of PIs in the defense response of plants to piercing-sucking insects remains questionable though, since these insects appear to lack endoproteinase activity within their digestive tract (Rhabé et al., 1995). However researchers have reported the presence of proteases in aphid alimentary tract and saliva (Bramstedt, 1948; Srivastava and Auclair, 1963; von Dehn, 1961; Klingauf, 1987). Van Emden (1966) studied the factors of resistance in plants to Myzus persicae and concluded that this aphid benefits in particular from the products of proteolysis in the plant. Many researchers have attached importance to the availability of amino acids to the aphids' feeding site since this determines growth and reproduction.
Poehling and Morvan (1984) and Poehling and Doerfer (1984) reported that only small amounts of amides and amino acids are likely to be translocated from other tissues to the feeding sites of aphid colonies and that this increase is mainly the result of synthesis and/or lysis of leaf proteins. This observation supports our findings that the infestation of A. fabae introduced a change in the concentration of individual amino acids (Figure 23). The remarkable aspect of our results is that the concentration of serine in supernatant of B. subtilis strain FZB37 induced plants remained the same prior to and post A. fabae infestation. Other individual amino acids, besides serine, may have played a role in the observed antibiosis effect but research has not yet been conducted to determine to what extent this may be the case. Other individual amino acids whose concentrations changed after supernatant treatment compared to the control (water treatment) were cysteine, alanine, tyrosine, and leucine.
Weismann and Halanda (1968) attributed the development of A. fabae on spindle, Euonymus europaeus, to the presence in high concentration of asparagine and arginine, which accords with our findings. The asparagine level in our research was very low after supernatant treatment, when compared to culture filtrate and control treatments. The higher concentration of alanine, reported as phagostimulant, observed here in the supernatant of B. subtilis strain FZB37 treatment, when compared to the control and culture filtrate of B. subtilis strain FZB37 treatments, could not have overturned the antibiosis effect inflicted by the inducer to A. fabae.
Chlorophyll fluorescence is very useful in the study of the effects of biotic and abiotic stress on plants since photosynthesis is often reduced in plants experiencing adverse conditions, such as water deficit, temperature, nutrient deficiency, polluting agents, attack by pathogens and insects (Fracheboud, 1999). In our study the chlorophyll fluorescence yield in treated V. faba and T. aestivum plants with supernatant of B. subtilis strain FZB37 and infested with A. fabae and R. padi respectively, was found to be higher, though not statiscally significant (Tables 24, 26), compared to the control (water treated plants). The reduced aphids’ fresh weight and dry weight (Tables 25, 27) in supernatant treatments could be an indication that the tested insects’ biology has been affected as statistically verified in the case of A. fabae treatment (Table 25).
Aphid feeding is often associated with a creation of a pathological sink, which concurs with the plant natural sink and reduces infected plants' photosynthesis capacity to a low level. In our study, after six days of sucking activity, A. fabae and R. padi feeding on plants treated with B. subtilis metabolites failed to fully reorganize their host plants' nutrient for their own nutritional needs. Wang et al. (2004) who used photosynthetic-rate measurement to assess resistance of wheat plants through aphid and plant biomass analysis reported that the biomass of D. noxia feeding on susceptible wheat plant was significantly higher than the one of D. noxia from resistant wheat lines. Similar observation was made in our research. Due to the small number of replications used in the experiments, it was not possible to confirm this result statistically.
Seed pre-treatment with spore suspensions showed a higher and significant chlorophyll-fluorescence yield of T. aestivum seedlings infested with R. padi (Table 30). Fresh weight and dry weight (Table 31) of R. padi caged on treated plants were diminished compared to the control plants (water treatment). The biology of the R. padi was negatively affected as it has failed to establish a pathological sink detrimental to the plants pre-treated with spore suspensions of B. subtilis. The negative effect of aphid attacks on plants was reported by Mallott and Davy (1978), who stated that two weeks of sucking activity by R. padi led to a diminished vegetative growth of barley as well as a reduced photosynthesis capacity. In his research, Witmann observed that fresh mass of leaves from induced plants that were submitted to aphid attacks had higher weight compared to untreated plants. In field tests potato and cotton seeds pre-treated with B. subtilis spore suspensions have demonstrated better yields compared to control plants treated with conventional fertilizers (Yao and Bochow, 2002; 2003).
Relevant literature reports, in addition to the positive effect of better growth attributed to spores suspensions of B. subtilis, some cases of foliar disease and insect control via seed treatment with induced resistance agents. Field experiments in cucumber demonstrated that plants grown from seed treated with PGPR sustained significantly lower populations of cucumber beetles, Diabrotica undecimpunctata howardi and Acalymma vittatum, as well as a lowered incidence of bacterial wilt disease when compared with nontreated control plants and plants sprayed weekly with the insecticide esfenvalerate (Zehnder et al., 1997). The findings of these researchers also demonstrate that PGPR reduce the triterpenoid beetle-feeding stimulant cucurbitacin. The direct benefit of PGPR to crops is manifold. Press et al. (1996) demonstrated the capacity of some genera of PGPR bacteria to induce systemic disease resistance in crops by producing salicylic acid. In our experiment, the higher chlorophyll-fluorescence level could be attributed to the PGPR qualities of B. subtilis, which have already been discussed in numerous other studies (Doley and Bochow, 1996).
Interestingly, it was found in our research that the supernatants of the three different strains of B. subtilis (FZB24, FZB37 and FZB38) used could induce resistance in both V. faba and T. aestivum to A. fabae and R. padi respectively. The inducer could also reduce the development of urediospores produced by Uromyces appendiculatus. This supports the hypothesis of the unspecific character of the inducer postulated in the introduction and concurs with the findings of others. B50-treated wheat and barley have been demonstrated to be resistant against Erysiphe graminis (Schönbeck et al., 1982; Kehlenbeck et al., 1994). Foliar jasmonic acid applied at concentrations not causing toxicity significantly reduced the performance of Tetranychus pacificus as well as the root-feeding grape phylloxera (Viteus vitifoliae) in grapevines (Omer et al., 2000). By inoculating tobacco leaves with tobacco mosaic virus, reproduction of Myzus persicae was reduced by 13 % and the growth rate of Manduca sexta was reduced by 27 % on the treated plants and resistance to viral, bacterial and pathogens was increased (McIntyre et al., 1980). In the same vein, Agrawal (1998) also reported that induced resistance observed by him was not species-specific and affected earwigs, aphids, grasshoppers, flea beetles, and lepidopteran larvae.
However a cross-protection function of the inducer is not always the case. Thus Galler et al. (1998) reported that B50-treated wheat is resistant against Sitobion avenae but not against Rhopalosiphum padi. Specificity in induced resistance can be distinguished in two ways. Firstly, by a range of organisms being affected by a given response trigger by the inducer, or secondly, by the capacity of the induced plant to generate distinct chemical responses to different types of damage. If a given inducer possesses these two major qualities, it is very probable that this inducer's field of activity is larger. In our study our inducer demonstrated its capacity to challenge a biotrophic pathogen and two different species of aphids. Nevertheless, further studies are necessary to determine the type of secondary metabolites produced by the treated plants infested with the pathogens and pests, as plant response to pathogens or pests can be type-specific. Specificity elicitation showed that aphid feeding induced peroxidase and lipoygenase activities but not polyphenol oxidase (catechol oxidase) and proteinase inhibitor activities, as opposed to Helicoverpa zea whose feeding induces polyphenol oxidase and proteinase inhibitors but not those of peroxidase (Stout et al., 1998). Furthermore, Agrawal (2000) observed that specialist and generalist herbivores may also respond differently to induced plant responses and in some cases induced phytochemicals that inhibit feeding by some herbivores are feeding stimulants or toxins sequestered by specialists (Agrawal and Karban, 1999). Carroll and Hoffman (1980) here showed that induced cucurbitacins in Cucurbita moschata attracted some herbivores while inhibiting feeding by others. However, it is also emphasised in the literature that this interreaction between insect response and plant secondary compounds is complex. Hence it should be noted that although specialist herbivores may be attracted to plants with high levels of secondary compounds (Bowers, 1992), they may still be susceptible to the toxic effects of the phytochemicals (Adler et al., 1995). Thus the investigation of plants secondary metabolites is of great interest for better performance of B. subtilis metabolite induced plants.
Attempts to reduce crop damage have included selective breeding for resistance, but often many different genes control resistance traits, making it difficult or even impossible to genetically select a desired attribute. Decreased crop yields are also commonly encountered in resistance strains. Accordingly, there exists a strong need for compositions and processes to improve the resistance of plants under attack by herbivores (Ryan, 1995). It would be of great interest to study qualitatively the supernatant and the culture filtrate of B. subtilis in order to identify the main substance(s) in the supernatant, responsible for the transduction of signals necessary for the activation of the plant defense system. Once such information is available, the supernatant can perform better and be used as a stable bioproduct. This constitutes an important step towards the production of the supernatant of B. subtilis as a commercially-available induced resistance agent.
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