2.  Material and methods


2.1.  Soil substrate

The soil substrate for plant cultivation in the leaf treatment experiments was a pure commercial substrate “Frühstorfer Einheitserde”, Type P. Part of this soil substrate was sterilised using a steamer machine “STERILO 1k” at 80°C for 12 hours and was used in the seed treatment experiment after cooling.

2.2. Plant material


For the purpose of these experiments, monocotyledon and dicotyledon plants were used.

2.2.1.  Broad bean (Vicia faba) cv. ‘Hangdown’

Vicia faba seeds were sown in plastic multiple quick pots and, when sprouting, were transferred into 12 cm diameter pots filled with 600 ml of soil substrate. The plants were stored in greenhouse conditions or in a growth chamber, under 16:8 h photoperiod light. The temperature in the greenhouse was 20°C ± 4°C and the relative humidity was between 50-80 %. In the growth chamber, the temperature was 20°C ± 2°C and the relative humidity was between 60-70 %. The plants were regularly watered with tap water and plants of approximately the same size were selected for the planned experiment. All plants used in the experiment unit were aged between two and four weeks.

2.2.2. Summer wheat (Triticum aestivum) cv. ‘Nandu’

The wheat seeds were grown under the same conditions as for Vicia faba (described above), except that instead of one plant per pot, four plants were grown in each pot.

2.3. Test organism breeding conditions

2.3.1.  Fungi


Bean rust (Uromyces appendiculatus) - spores of Uromyces appendiculatus isolate SWBR I were kindly provided by Prof. Kurt Mendgen, University of Konstanz (Germany). The spores were stored at –20°C to preserve the germination capacity prior to use in the test.

2.3.2.  A. fabae and R. padi breeding conditions

Stock cultures of A. fabae and R. padi were placed on Vicia faba and Triticum aestivum plants, respectively and maintained in the growth chambernder 20°C, 60-65 % relative humidity and light/dark regime of 16/8 hours.

The apterous of A. fabae and R. padi used in all greenhouse experiments were standardised in the following procedure: apterous of A. fabae and R. padi were caged independently on Vicia faba and Triticum aestivum plants and after 24 hours, solely the offspring were kept; these offspring were then allowed to continue their development under the conditions described above until adulthood. The standard insects thus obtained were then used in the experiments.

2.4. Clip cages used in aphid tests


The clip cages used in these experiments were identical to those described by Noble (1958) and slightly modified versions of those used by the Poehling working group in IPP, Univ. Hannover. The border of the Petri dish, with a 35 cm diameter, was brought into contact with an acetone solution for a few seconds, after which a ring of rubber was placed over and fixed to it. This ensured the clip cage was machine washable without any deterioration of the assembled materials.

Figure 1:Clip cages fixed on test plant leaflets; right and left on Vicia fabae and Triticum aestivum leaflet, respectively

2.5.  Bacillus subtilis and its metabolites

Bacillus subtilis strains, FZB24, FZB37 and FZB38 and its metabolites were provided by the laboratory FZB Biotechnik GmbH, Berlin. The culture filtrate of B. subtilis isolate B50 used in these experiments was kindly provided by the IPP Univ. Hannover.

2.5.1.  Biology and morphology of Bacillus subtilis (Ehrenberg) Cohn


According to its morphology and biology, Bacillus subtilis belongs to the Bacillus species, the Bacillaceae family and the Eubateriales order (Mueller, 1965; Jacob et al., 1981). It was first described by Ehrenberg (1835) and later by Cohn (1872). The bacterium has a stick form and peritric flagella, is gram positive, aerobe, and is a spore-forming bacterium (Schlegel, 1992). The temperature interval of the bacteria is 5-55°C (Sinclair, 1989) with an optimum at 25°C (Gupta and Utkhede, 1986). The pH of the bacteria is between 4.5 and 8.5 with the optimum value around 6-7.5 (Thimann, 1964). Soil is the reservoir of this bacterium, from where it is transferred to various associated environments including plants and plant materials, foods, animals and marine and freshwater habitats (Priest et al., 1987).

Bacillus subtilis is capable of producing peptide-type antibiotic during its fermentation phases, which are respectively logarithmic, transition and stationary (McKeen et al., 1986). Several strains of B. subtilis are cited as producing cyclic lipopeptides, which belong to the family of iturin, fengymycin (Besson et al., 1978; Peypoux, 1980; Mhammedi et al., 1982; Loeffler et al., 1990; Asaka and Shoda, 1996a; Hbid et al., 1996) and surfactin group. The B. subtilis/amyloliquefaciens group has been reported to produce IAA and phytase as a phosphorus-mobilizing enzyme essential for plant nutrition. Idris et al. (2002) reported that B. subtilis strains, FZB24, FZB42 and FZB45 showed extra cellular phytase activity when cultivated in wheat bran extract, which is known to contain phytate.

2.5.2.  Bacillus subtilis metabolite productions

After inoculation of Bacillus subtilis spores in the Landy-Medium, a lag phase commences during which the cells increase in mass but do not divide. This is then followed by a phase of exponential growth (also called transition phase) whose physiological state is marked by back-to-back division cycles such that the population doubles in number every generation. During the exponential growth there is no change in average cell mass. Although the cells constantly change in mass as they increase, they then divide rapidly, decreasing in mass. As the rate of growth is exponential, the rate of increase in cell number is initially slow but increases at an ever-faster rate, resulting in the later stages in an explosive increase in cell numbers (Madigan et al., 1997). The exponential phase is followed by the stationary phase classically defined as a physiological point where the rate of cell division equals the rate of cell death; hence viable cell numbers remain constant. The stationary phase usually occurs when cell concentration is so great that an aspect of the environment is no longer able to serve the requirements of exponential growth. Generally, either an essential nutrient of the culture medium is exhausted or some waste product of the organism builds up in the medium to an inhibitory level and exponential growth ceases (Madigan et al., 1997). The stationary phase is a time of significant physiological change and particularly involves the physiological adaptation of cells to survival through periods of little growth (Abedon, 2001).


During this development period, B. subtilis is understood to produce different kinds of metabolites as enzymes and lipopeptide antibiotics (Doley, 1998) which cause important qualitative and quantitative changes in the substance spectrum. During the logarithmic phase, B. subtilis has been noted as producing hydrophile oligopeptide antibiotic as bacilysin (Walker and Abraham, 1970; Hilton et al., 1988; Loeffler et al., 1990; Koumoutsi et al., 2004), chlorotetain and rhizocticin (Loeffler et al., 1990), while the stationary phase was characterized by the production of lipopeptide antibiotic as iturin, fengymycin, and surfactin type as the cyclopeptide mycobacillin (Loeffler et al., 1990; Besson, 1994; Ohno et al., 1995).

Figure 2 shows the laboratory principle of production of B. subtilis metabolites using Landy – Medium (Table 1).

Figure 2: Diagram of the production of Bacillus subtilis and its metabolites. CF: Culture Filtrate; sup: supernatant; vc: vegetative cells; sp: spore suspensions


Table 1: Composition of the Landy-Medium (LM) for 1 liter of solution



MgSO4 (pure)




Glutamic acid* (pure)


MnSO4 .H2O






* The marked elements were separately diluted and sterilised.  Spore suspensions

The spores used in this experiments were obtained after 72 hours of cultivation, and then washed from the medium by centrifugation at 9000 g/min. Prior to the experiment, the necessary dilution was prepared and the obtained bacterial spore suspensions were used in the activated form by placing them in a warm bath at 60°C for 15 min and then cooling them under laboratory conditions. Culture filtrate

The culture filtrate corresponding to each of the different bacterial growth phases, logarithmic, transition and stationary was obtained by passing the culture supernatant through a Millipore filter (0.2 µm) into sterile flasks. The characteristics of culture filtrate, strain FZB24 of B. subtilis in Landy-Medium (Table2) were determined by Beckmann (1995) and FZB Biotechnik GmbH (1995).


Table 2: Characteristics of culture filtrate of strain FZB24 of Bacillus subtilis in Landy-Medium (Beckmann, 1995 and FZB Biotechnik GmbH, 1995)



7 hours

trans. phase

12 hours

stat. phase

72 hours





Osmotic pressure





Glutamic acid










NH4 (µg/ml)









Cyclic lipopeptide




0.242 Supernatant

The supernatant of Bacillus subtilis isolate FZB24, FZB37 and FZB38 was used in the transition phase after centrifugation of fermented bacteria. Vegetative cells

After 24 hours of fermentation at 34°C, and following 10 min of centrifugation at 9000 g/min, the vegetative cells were separated from the supernatant, suspended in a physiological salt solution and homogenised.

2.6. Utilisation of the obtained materials in the experiments

2.6.1.  Seed pre-treatment with spore suspensions prior to sowing  Treatment procedure


The seed materials were first disinfected with sodium hypochlorite (3% active Cl) in aqueous solution for 10 min and rinsed three times with sterile distilled water. The seeds were left to dry and then dipped for 10 min in water, which contained 105 cfu/ ml of B. subtilis spore suspensions. The control seeds were dipped in distilled water and after a short drying period, they were sown into sterile soil in multiple quick pots and later transplanted into 12 cm diameter pots filled with sterile commercial substrate, as described above. Each pot was watered with the same quantity of distilled water. Reisolation of Bacillus subtilis from the roots

For the reisolation of B. subtilis from the rhizosphere, 1 g root (fresh weight), according to each variant of the treatment, was taken separately from composite samples of 5 Vicia faba plants and 4 wheat plants. The samples were shaken in 99 ml sterile sodium chloride-solution (0.3%) at 220 rpm for 20 min. The dilution series were then prepared and 100 µl from each dilution was mixed with cooled agar in petri plates. The colonies were expressed as the number per gram of roots by measuring the total weight of dry matter from each homogenate. After two days incubation at 25°C, the number of B. subtilis cfu per g root and substrate dry weight were measured, respectively. Here no attempt was made to differentiate the spores from the vegetative cells in the counting method utilised.

2.6.2. Application of spore suspensions and vegetative cells on the leaves

The spore suspensions 107 cfu/ml and vegetative cells 107 cfu/ml were applied separately to Vicia faba plants, including the leaves, which were completely sprayed using a hand sprayer. After the foliage dried out, the standardised apterae of A. fabae were caged on the selected leaves.

2.6.3. Topical treatment of the plant leaves using culture filtrate and the supernatant


The culture filtrate and the supernatant, each a 10 % solution, were sprayed on the plants, including the leaves, using a hand sprayer. After a period of 2-3 days, the standardized insects were caged on the leaves selected for the aphid tests.

2.6.4. Systemic treatment of the plant leaves using culture filtrate and the supernatant

In order to assess the supposed systemic effect of B. subtilis metabolites, the two first leaves of V. faba plant (counting upwards from the bottom) and the corresponding part of the stem were isolated from the rest of the plant and sprayed with the test solutions. This was to asses whether the metabolites of B. subtilis have only a local effect or, in fact, a systemic one, i.e. whether the effect of metabolites could be transported through the other organs of the plant when treatment is limited to a certain part of the plant.

Figure 3: Systemic induced treatment of a Vicia faba plant.

The three first leaves of Vicia faba plant and corresponding stem, marked white in the photo, were isolated and treated with Bacillus subtilis metabolites in a systemic test.

2.6.5. Application of supernatants of Bacillus subtilis in acute toxicity test


In order to determine whether the supernatants have a direct contact effect on A. fabae, an acute toxicity test was conducted. For this purpose, 2 ml of supernatant of B. subtilis per treatment was poured over a filter paper placed in a petri plate. The test insects were then gently placed on the wet filter paper for 5 minutes and then removed. In the case of the control insects, water was poured over the filter paper. The insects were then allowed to dry and were then caged onto the untreated plants.

2.7. Rearing Aphis fabae on sterile synthetic diet

Table 3: Composition of synthetic diet (mg/ 100 ml of diet)




15 000.0



Ascorbic acid





Aspartic acid




Cysteine HCL






Nicotinic acid







free base




Folic acid




Ca pantothenate






Lysine HCl


Choline chloride
















Fe sequestrenea




Zn sequestrene




Mn sequestrene




Cu sequestrene




Formulation: pH adjusted to 7.0 with KOH 1.75 M, and water (distilled-deionised) to make
100 ml of diet.
a Sequestrenes are compounds of the metal with sodium EDTA.

2.7.1.  Synthetic diet preparation procedure

The composition of the diet, shown in Table 3, accorded with the specifications of Dadd and Krieger (1967). The diet was prepared in volumes of 500 ml, as described by Akey and Beck (1971). The amino acids, vitamins, and salts were added to a sucrose solution of ca. 2/3 of the desired total volume and the mixture was stirred with a counter-rotating mixer for 1-2 hours under a stream of nitrogen gas. The ferric, zinc, and manganese sequestrene were made up as individual stock solutions and added to the diet at the end of the mixing period. The solution was adjusted with 1.75 M KOH to a pH of 7.0, then increased to the final volume and finally passed through a Millipore filter (0.2 µm) into sterile flasks. The diet was stored in 25 ml quantities at –20°C for up to 1 month.

2.7.2. Feeding apparatus used in sterile feeding test


The experiment cages were composed of a plastic petri plate, 2 rings of equal size, one of which had an incision on the side, and two parafilm layers. Under a laminar box, 2 ml of sterile diet was first poured over one parafilm layer placed over the intact ring. The second layer of parafilm was used to cover the deposited liquid. Care was taken that the liquid did not overflow. Finally, the second, incised ring was stretched over the first ring and the two layers of parafilm, thereby sandwiching the diet between the two pieces of parafilm.

Figure 4: Feeding apparatus for artificial diet. Left photo: two rings with the one ring cut on the side. Right photo: the two layers of parafilm with the feeding liquid sandwiched between them. The ring with the incision is stretched over the intact ring and the two layers of parafilm.

In order to maintain sterile and aseptic conditions during the feeding procedure of the aphids, distilled water was autoclaved for 20 min at 121°C and the parafilm membranes were kept in 70 % ethanol for approximately 24 h, following the methodology of Akey and Beck (1971).

2.8. Artificial infestation of Vicia faba with Uromyces appendiculatus


Urediospores of Uromyces appendiculatus were added to a solution of distilled water containing 0.01 % Tween-20 and stirred with a magnetic stirrer for 5 min. Urediospore concentration was determined using a Bueker haemacytometer and adjusted to 100 000 spores per ml. The obtained suspension was sprayed onto the leaves of two-week-old plants of Vicia faba that had been pre-treated 2-3 days prior with B. subtilis culture filtrate, supernatants and Landy-Medium. The control plants were pre-treated with water. After the film dried, the plants were incubated at 100% r.h. in the dark. The container was then closed and stored in darkness, which were required conditions for the Uromyces appendiculatus to be active. The plants were removed 24 hours later and placed in the growth chamber. After 8-14 days, uredial pustules had developed and were counted.

2.9. Life table tests for Aphis fabae and Rhopalosiphum padi

To determine the relative growth rate (RGR) and the intrinsic rate of natural increase (rm), life table model experiments were carried out.

2.9.1.  The estimation of Relative Growth Rates of Aphis fabae and Rhopalosiphum padi

Seedlings of Vicia faba and Triticum aestivum, when aged 3-4 weeks, received one standardised adult aptera of Aphis fabae or R. padi respectively, on selected leaves. The aptera was fixed onto the leaves with a clip cage. After 24 hours, 50 new-born larvae (L1) were collectively weighed to determine the mean weight (A) in mg, according to each variant of the experiment and, of these, a single larva was kept. When the insects performed their development within time (tD) in days (Fischer, 1921; van Emden, 1969), i.e. the time from birth to adult moult, the individual weight (B) in mg of each insect was again recorded. All weight measurements in the aphid tests were taken using a fine-balance Sartorius MC5 with readability from 1 µg up 5g.


The three parameters, (A), (B) and (tD) are used to calculate the RGR, which is the growth per unit weight per unit time of an aphid (Fischer, 1921; van Emden 1969):

The concept of relative growth rate is familiar to plant physiologists. Radford (1967) points out that the use of the RGR does not involve any assumption about the form of the growth curves, and is therefore particularly valuable for comparing different results of different treatments.

2.9.2. The e estimation of the intrinsic rates of natural increase (rm) of Aphis fabae and Rhopalosiphum padi


The estimation of rm was carried out according to Wyatt and White (1977). Acordingly, the pre-reproduction time (td), corresponding to the day when young larvae in the experimental group were first observed and marked, and the number of larvae born per adult per day were counted during a period of time equalling 2 × (td). To avoid double counting, the already counted larvae were immediately removed from the experiment. During this period of time, 2 × (td), the recorded number of larvae (Md) served as effective lifetime fecundity, corresponding to the number of larvae born in a generation time (td). Based on Md and (td), the equation of rm is postulated theoretically by Dixon (1987) as the rate of increase of a population that has assumed constant age schedule of births and deaths and increasing in number in an unlimited space. The formula is given as follows:

This above equation has been used by several researchers to calculate the rm value for aphids (Leather and Dixon, 1984; Sandström, 1994; Sandström and Pettersson, 1994; Soraka and Mackay, 1991; Birch and Wratten, 1984; Sotherton and van Emden, 1982). In the formula, k serves as a correcting constant with a value of 0.754 and 0.745 (Frazer, 1972) for A. fabae and R. padi,respectively. The rm value derived from this equation is probably more useful in laboratory assessment than in the field. As Carter et al. (1980) pointed out, aphid populations rarely if ever achieve a stable age for distribution and therefore rm represents a limited value in the field. The experiments mentioned here were conducted in the greenhouse under a controlled environment at 25°C ± 2°C.


To assess the life table of A. fabae and R. padi, the plant species, V. fabae (1 plant per pot) and T. aestivum (4 plants per pot) were each arranged in 20 replicates, in random blocks.

2.10. Physiological tests

2.10.1.  Chlorophyll fluorescence measurement

The physiological status of green plants can be determined by measuring chlorophyll fluorescence. The level of chlorophyll fluorescence and its spectral distribution depends on a number of factors related to the ability of a plant to perform photosynthesis, which in its turn is dependant on adequate plant-growth conditions. Plants stressed by biotic and abiotic factors probably limit chlorophyll production and exhibit both a lower overall level of fluorescence and a shift in spectral distribution, compared to healthy plants.

To assess this parameter in this study, a fluorimeter PAM-2000 (Heinz Walz GmbH, Effeltrich, Germany) was used to investigate intact selected plant leaves, which had been pre-treated with B. subtilis and its metabolites. Water-treated plants served as control. The intact leaf to be measured was inserted into the leaf clip of the fluorimeter. The system then automatically switched on the measuring light and with every saturation pulse the measured data, which was calculated online, was written into a report–file. Fluorescence is excited by very brief but strong light pulses from light-emitting diodes. With the PAM-2000, these pulses are 3 µsec long and repeated at a frequency of 600 or 2000 Herz. The LED light passed through a short filter (λ<670 nm) and the photodetector was protected by a long-pass filter (λ>700 nm), as well as a heat-absorbing filter. A highly selective pulse amplification system ignored all signals except the fluorescence excited during the 3 µsec measuring pulses. The photodetector was a PIN-photodiode displaying linear response, while the light intensity changed by factors of more than 109. Hence, this measuring system tolerated extreme changes in light intensity (up to several times the intensity of full sunlight) even at light intensities that measured weakly. All yield measurement experiments were performed under cell conditions at 20°C ± 2°C. The laboratory facilities used were provided by Prof. Schmitt J. at the Freie Universität Berlin, FB Pflanzenphysiologie.


Figure 5: Chlorophyll fluorescence measurement of Triticum aestivum leaf, using a fluorimeter PAM-2000

2.10.2. Amino acids investigation

The investigation of amino acids aimed at determining any quantitative and qualitative change following the treatment of V. faba plants with B. subtilis metabolites.  Sample collection

When aged between 3–4 weeks, Vicia faba seedlings treated with B. subtilis metabolites and control (water-treated) seedlings received on their fourth leaves (counting upwards from the bottom) one clip cage, containing 6 adults of A. fabae for each seedling. After a 6-day sucking process, 0.2–0.3 g of the youngest leaves from each seedling were harvested, placed on aluminium sheets and immediately introduced into liquid nitrogen. All samples were collected between 10 a.m and 12 a.m and placed in a freezer at a temperature of –50°C prior to extraction. The experiment was arranged for treated and control plants in two groups: treated V. faba plants, with clip cages containing insects, and parallel plants with empty clip cages. The empty-clip-cage approach sought to eliminate any effect this assemblage may have in the experiment. Amino acids extraction and analysis


The samples were first dry frozen (-20°C, 0.06 mbar, minimum 2 days) and were then thawed to a constant weight, with a deviation of no more than 1 %. The dry material was ground in a mortar; 35 mg of the obtained fine powder was weighed in a 1.5 ml Eppendorf capsule.Each sample had 1000 μl of 80 % ethanol and then 5 μl of internal standard [s-carboxymethyl-l-cysteine] added before being placed in an ultrasonic bath (50-60 KHz, ice cool) for 10 min. The solution was then centrifuged 3 times at 4°C, 6000 g for 10 min each time. During the second and third times, 700 μl of 80% ethanol and 300 μl of 80 % ethanol respectively were added to the debris. Finally, the combined 80 % ethanol supernatant was gathered in a 2 ml Eppendorf capsule. From this, 150 μl was introduced in a millipore ultra-free filter and centrifuged at 4°C, 5000 g for 1–4 hours in order to separate the proteins. Finally the filtered solution was used for the amino acid test.

The amino-acid analysis was performed on the modular Knauer Amino Acid Analyser 830, consisting of 2 HPLC pumps, Type 64, equipped with micro-pump heads, the derivatizer-autosampler, the gradient programmer 50 B, a dynamic mixing chamber, a high temperature oven with temperature control unit, the fluorescence spectromonitor RF 535 or the variable wavelength detector, and the data processor chromatopac C-R6A.

The separation of the derivatised amino acids was performed on Knauer OPA with analytical (250 x 4 mm) or narrow bore (250 x 2 mm) size.


The OPA analytic method (Roth, 1971; Godel et al., 1984; Nakazawa and Arima, 1982; Ashman and Bosserhoff, 1985) was used for the amino-acid analysis.

The amino acids were derivatised automatically using the Knauer Derivatizer-Autosampler at 25°C. The OPA reaction mixture was prepared from 100 μg OPA, 1000 μl methanol, 60 μl mercaptoethanol and 9 ml 1 M boric acid pH 10.4. This mixture was stored frozen at — 20°C and freshly prepared each week.

The amino acids were separated in a gradient from buffer A (90 % 12.5 mm di- sodium hydrogen phosphate pH 6.5 and 10 % methanol) to buffer B (methanol and 3% v/v tetrohydrofuran). The detection was performed using the fluorescence detector at Ex 340 nm, Em 455 nm. Amino-acid standards or analysed samples were diluted in a citrate buffer (19.6 g sodium citrate x 2 H2O, 20 ml thiodiglycol, 30 ml mercaptoethanol filled with water up to 1000 ml, pH 2.2).


The reaction was performed at 25°C, and the reagent-sample v/v ration was 1:1 (10 μl). OPA and all other reagents were analytical grade from Merck (Darmstadt, Germany).

The above-mentioned acid analysis was performed under the supervision and guidance of Dr. Godt Franz at the Technische Fachhochschule, Berlin.

2.11. Statistical analysis

The statistic analyses were carried out as outlined by Koehler et al. (1984) and Buehl and Zoefel (1998). The various experiments discussed here were repeated more than twice. The numbers of replications are documented below, in Chapter 3. The data of the life tables were tested to determine if assumptions of homogeneity of variances were correct. To test the null hypothesis that B. subtilis and its metabolites treatments can negatively influence the growth parameters of A. fabae and R. padi, SPSS ANOVA Tukey multiple analysis testing was used (P< 0.05). In each experiment group, the samples were independently and randomly arranged to minimise systematic error. The statistically significant levels in each group of experiments are marked with different alphabetic letters (see tables and graphics).

© Die inhaltliche Zusammenstellung und Aufmachung dieser Publikation sowie die elektronische Verarbeitung sind urheberrechtlich geschützt. Jede Verwertung, die nicht ausdrücklich vom Urheberrechtsgesetz zugelassen ist, bedarf der vorherigen Zustimmung. Das gilt insbesondere für die Vervielfältigung, die Bearbeitung und Einspeicherung und Verarbeitung in elektronische Systeme.
DiML DTD Version 4.0Zertifizierter Dokumentenserver
der Humboldt-Universität zu Berlin
HTML generated: