3. Results

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3.1.  Influence of Bacillus subtilis metabolites on Uromyces appendiculatus, Aphis fabae and Rhopalosiphum padi

3.1.1.  Uromyces appendiculatus

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Bacillus subtilis metabolites were assayed against Uromyces appendiculatus to gain an indication of the capacity of the isolates used in this research to inhibit the development of the pathogen urediopustules.

In topical and systemic treatment of the host plants (Table 4), the supernatants of B. subtilis strains FZB24, FZB37 and FZB38 and the culture filtrate of strains FZB24, FZB37 and FZB38 were able to reduce the number of uredial pustules produced by Uromyces appendiculatus compared to the control (water-treated) and the Landy-Medium (LM).

An inhibitive effect was not observed on U. appendiculatus spores development for either V. faba or the control (water treatment) seedlings in the Landy-Medium treatment.

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Table 4: Effect of Vicia faba seedlings’pre-treatment with Bacillus subtilis metabolites on the number of rust pustules produced by Uromyces appendiculatus

Treatment

Topical

(number of pustules per leaves)

Systemic

(number of pustules per leaves)

Control

965 ± 338.8 a

1111 ± 404.3 a

Landy - Medium

873 ± 325.7 a

822 ± 314.8 a

FZB24 sup

80 ± 33.2 b

275 ± 98.6 b

FZB37 sup

66 ± 23.8 b

341 ± 95.0 b

FZB38 sup

75 ± 26.9 b

262 ± 125.0 b

FZB24 CF

344 ± 118.9 b

FZB37 CF

336 ± 121.5 b

FZB38 CF

417 ± 51.9 b

Values within the same column are means ± standard deviations (n = 10 for FZB24 CF, FZB37 CF, FZB38 CF; n = 5 for control, LM, FZB24 sup, FZB 37 sup, FZB38 sup) and those flanked by different letters are statistically significant (Tukey’s test; p< 0.05).

Figure 6: Uromyces appendiculatus development on pre-treated Vicia faba seedlings with Bacillus subtilis metabolites. Left: control (water treatment); middle: systemic treatment with supernatant FZB37; right: topical treatment with supernatant FZB37

3.1.2. Influence of pre-treatment of seedlings of Vicia faba with culture filtrates of B. subtilis FZB24, B50 and Landy-Medium on feeding of Aphis fabae

The application of culture filtrate of the Bacillus subtilis strain FZB24 obtained from the three different fermentation phases, logarithmic, transition, and stationary, could not reduce the growth parameters of A. fabae compared to the control plants treated with tap water (Tables 5, 6). The same result was found after the usage of Landy-Medium and B50 against A. fabae. While treatment with culture filtrate of B. subtilis at the transition phase showed the lowest rm, among the treatments, this was not statistically verified (Tables 5, 6).

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Table 5: Influence of culture filtrate of Bacillus subtilis FZB24 at logarithmic, transition and stationary phases, Landy-Medium and B50 on the growth parameters of Aphis faba feeding on Vicia faba

Treatment

A

(mg)

B

(mg)

tD

(days)

RGR

Control

0.036

0.613 ± 0.140 a

6.1 ± 0.3 a

0.461 ± 0.049 a

LM

0.037

0.600 ± 0.152 a

6.1 ± 0.3 a

0.5 00 ± 0.055 a

Log

0.035

0.617 ± 0.120 a

6.1 ± 0.2 a

0.471 ± 0.042 a

Trans

0.038

0.629 ± 0.144 a

6.2 ± 0.4 a

0.453 ± 0.050 a

Stat

0.045

0.631 ± 0.146 a

6.2 ± 0.4 a

0.426 ± 0.049 a

B 50

0.039

0.566 ± 0.134 a

6.2 ± 0.4 a

0.430 ± 0.049 a

Values within the same column are means ± standard deviations (n = 20 for control, LM, log and Trans treatments; n= 12 for Stat.; n= 8 for B50) and those flanked by different letters are statistically significant (Tukey’s test; p< 0.05). This experiment was performed in Hannover IPP Univ. under greenhouse conditions.
A: Average mean weight of newly born insect larvae (L1); B: Adult weight of individual insect; tD: development time, time from birth to adult moult; RGR: Relative growth rate.

Table 6: Influence of culture filtrate of Bacillus subtilis FZB24 at logarithmic, transition and stationary phases, Landy-Medium and B50 on the growth parameters of Aphis faba feeding on Vicia faba

Treatment

Md

td

(days)

rm

Control

44.4 ± 9.6 a

7.6 ± 0.7 a

0.391 ± 0.028 a

LM

47.5 ± 9.5 b

7.7 ± 0.7 a

0.389 ± 0.031 a

Log

49.9 ± 9.9 b

7.5 ± 0.5 a

0.395 ± 0.034 a

Trans

44.0 ± 10.9 a

7.5 ± 0.5 a

0.377 ± 0.03 a

Stat

52.9 ± 10.1 b

7.8 ± 0.6 a

0.396 ± 0.024 a

B 50

51.2 ± 11.2 b

7.4 ± 0.5 a

0.400 ± 0.03 a

Values within the same column are means ± standard deviations (n = 20 for control, LM, log and trans treatments; n= 12 for Stat.; n= 8 for B50) and those flanked by different letters are statistically significant (Tukey’s test; p< 0.05). This experiment was performed in Hannover IPP Univ. under greenhouse conditions.
Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

3.1.3.  Influence of pre-treatment of seedlings of Triticum aestivum with culture filtrate of B. subtilis strains FZB24, FZB37 and FZB38 on feeding of Rhopalosiphum padi

The application of culture filtrate of B. subtilis strains, FZB24, FZB37 and FZB38 onto the leaves of summer wheat (Triticum aestivum) did not significantly influence the growth parameters of R. padi (Tables 7, 8). Though a slight negative effect on the growth parameters of R. padi was observed, this was not statistically verified. There were indications that in the treatments, the development time and the pre-reproduction time are longer, in contrast to the RGR and the rm, which are shorter, when compared to the control (water treatment) (Tables 7, 8).

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Table 7: Influence of culture filtrate of three strains (FZB24, FZB37, FZB38) of Bacillus subtilis at transition phase on the growth parameters of Rhopalosiphum padi feeding on Triticum aestivum

Treatment

A (mg)

B (mg)

tD

(days)

RGR

Control

0.040

0.638 ± 0.130 a

6.0 ± 0.0 a

0.460 ± 0.033 a

FZB 24 CF

0.040

0.660 ± 0.137 a

6.2 ± 0.5 a

0.455 ± 0.045 a

FZB 37 CF

0.037

0.608 ± 0.12 a

6.3 ± 0.7 a

0.447 ± 0.050 a

FZB 38 CF

0.038

0.612 ± 0.097 a

6.2 ± 0.4 a

0.453 ± 0.035 a

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20).
A: Average mean weight of newly born insect larvae (L1); B: Adult weight of individual insect; tD: development time, time from birth to adult moult; RGR: Relative growth rate

Table 8: Influence of culture filtrate of three strains (FZB24, FZB37, FZB38) of Bacillus subtilis at transition phase on the growth parameters of Rhopalosiphum padi feeding on Triticum aestivum

Treatment

Md

td

(days)

rm

Control

39.9 ± 7.1 a

6.8 ± 0.6 a

0.413 ± 0.039 a

FZB 24 CF

41.3 ± 10.4 a

6.8 ± 0.4 a

0.409 ± 0.032 a

FZB 37 CF

39.8 ± 11.4 a

7.0 ± 0.3 a

0.394 ± 0.045 a

FZB 38 CF

41.8 ± 110 a

6.8 ± 0.4 a

0.410 ± 0.049 a

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20).
Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

3.1.4.  Influence of pre-treatment of Vicia faba seedlings with culture filtrate of Bacillus subtilis strains FZB24, FZB37, FZB38, and supernatant of B. subtilis strain FZB37 on Aphis fabae feeding

The application of culture filtrate of B. subtilis strains FZB24, FZB37, FZB38 and supernatant of strain FZB37 revealed that only the supernatant of strain FZB37 on V. faba was able to significantly influence the development of A. fabae. The growth parameters of A. fabae, tD and td are longer, whereas the RGR and the rm, were found to be lower in the FZB37 supernatant treatment compared to the control (water-treated) and the culture filtrate treatment (Tables 9, 10). Thus the RGR and td values in the FZB37 supernatant treatment were statistically different to the control and culture filtrate FZB24, FZB37 and FZB38 treatments (Table 9). The rm value was found to be statistically different from FZB 38 CF and control but not from FZB 24 CF and FZB 37 CF treatments (Table 10). Following these observations, further experiments were performed to test the supernatants of other strains of B. subtilis.

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Table 9: Influence of culture filtrate of three strains (FZB24, FZB3, FZB38) and supernatant FZB37 sup of Bacillus subtilis at transition phase on the growth parameters of Aphis fabae feeding on Vicia faba

Treatment

A

(mg)

B

(mg)

tD

(days)

RGR

Control

0.051

0.878 ± 0.129 a

6.0 ± 0.0 a

0.472 ± 0.026 a

FZB 24 CF

0.046

0.886 ± 0.124 a

6.1 ± 0.2 a

0.486 ± 0.027 a

FZB 37 CF

0.046

0.846 ± 0.191 a

6.1 ± 0.3 a

0.476 ± 0.047 a

FZB 38 CF

0.049

0.798 ± 0.142 a

6.2 ± 0.4 a

0.454 ± 0.048 a

FZB 37 sup

0.047

0.772 ± 0.233 a

7.2 ± 0.4 b

0.385 ± 0.053 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20).
A: Average mean weight of newly born insect larvae (L1); B: Weight of adult of individual insect; tD: development time, time from birth to adult moult; RGR: Relative Growth Rate

Table 10: Influence of culture filtrate of three strains (FZB24, FZB3, FZB38) and supernatant FZB37 sup of Bacillus subtilis at transition phase on the growth parameters of Aphis fabae feeding on Vicia faba

Treatment

Md

td

(days)

rm

Control

50.6 ± 9.2 a

7.2 ± 0.4 a

0.413 ± 0.023 a

FZB 24 CF

44.3 ± 11.0 a

7.4 ± 0.5 a

0.386 ± 0.038 b

FZB 37 CF

43.6 ± 8.8 a

7.5 ± 0.6 a

0.380 ± 0.037 b

FZB 38 CF

44.4 ± 11.9 a

7.3 ± 0.4 a

0.392 ± 0.044 a

FZB 37 sup

44.7 ± 11.1 a

8.5 ± 0.7 b

0.336 ± 0.034 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20).
Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

3.1.5.  Influence of pre-treatment of seedlings of Vicia faba and Triticum aestivum with supernatants of Bacillus subtilis strains FZB24, FZB37 and FZB38 on feeding of Aphis fabae and Rhopalosiphum padi

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The pre-treatment of V. faba and T. aestivum plants with supernatants of the three strains FZB24, FZB37 and FZB38 of B. subtilis clearly showed that the growth parameters of A. fabae and R. padi feeding on corresponding host plants were significantly affected (Tables 11, 12, 13, 14). The insects referred to presented longer tD and td, as opposed to a lower RGR and rm (Tables 11, 12, 13, 14). The effects of the supernatants of B. subtilis constitute a typical case of induced resistance in Vicia faba and Triticum aestivum plants.

While the supernatants of the different strains of B. subtilis (FZB24, FZB37 and FZB38) used in this research could influence growth parameters of A. fabae and R. padi, no statistical difference among the three strains was observed (Tables 11, 12, 13, 14).

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Table 11: Influence of supernatants from Bacillus subtilis FZB24, FZB37 and FZB38 on growth parameters of Aphis fabae feeding on Vicia faba

Treatment

A

(mg)

B

(mg)

tD

(days)

RGR

Control

0.050

0.823 ± 0.177 a

6.0± 0.0 a

0.463 ± 0.066 a

FZB 24 sup

0.049

0.612 ± 0.177 b

6.7 ± 0.8 b

0.377 ± 0.066 b

FZB 37 sup

0.045

0.594 ± 0.169 b

6.6 ± 0.6 b

0.391 ± 0.068 b

FZB 38 sup

0.053

0.679 ± 0.157 b

6.6 ± 0.5 b

0.388 ± 0.055 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20).
A: Average mean weight of newly born insect larvae (L1); B: Weight of adult of individual insect; tD: development time, time from birth to adult moult; RGR: Relative growth rate

Table 12: Influence of supernatants from Bacillus subtilis FZB24, FZB37 and FZB38 on Aphis fabae feeding on Vicia faba

Treatment

Md

td

(days)

rm

Control

55.4 ± 6.9 a

7.0 ± 0.0 a

0.432 ± 0.013 a

FZB 24 sup

52.5 ± 11.7 a

7.7 ± 0.7 b

0.389 ± 0.043 b

FZB 37 sup

46.6 ± 13.9 a

7.6 ± 0.6 b

0.383 ± 0.058 b

FZB 38 sup

49.2 ± 10.6 a

7.6 ± 0.5 b

0.386 ± 0.039 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20).
Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

Table 13: Influence of supernatants from Bacillus subtilis FZB24, FZB37 and FZB38 on Rhopalosiphum padi feeding on Triticum aestivum

Treatment

A (mg)

B (mg)

tD

(days)

RGR

Control

0,038

0.516 ± 0.092 a

5.9 ± 0.4 a

0.444 ± 0.037 a

FZB 24 sup

0.037

0.393 ± 0.084 b

6.0 ± 0.0 a

0.390 ± 0.036 b

FZB 37 sup

0.035

0.364 ± 0.103 b

6.0 ± 0.0 a

0.385 ± 0.056 b

FZB 38 sup

0.041

0.446 ± 0.107 a

6.0 ± 0.0 a

0.393 ± 0.044 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20 for control, FZB24 sup and FZB37 sup; n = 15 for FZB38 sup).
A: Average mean weight of newly born insect larvae (L1); B: Adult weight of individual insect; tD: development time, time from birth to adult moult, RGR: Relative Growth Rate

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Table 14: Influence of supernatants from Bacillus subtilis FZB24, FZB37 and FZB38 on Rhopalosiphum padi feeding on Triticum aestivum

Treatment

Md

td

(days)

rm

Control

38.8 ± 6.6 a

6.8 ± 0.4 a

0.400 ± 0.028 a

FZB 24 sup

30.1 ± 6.1 b

7.1 ± 0.2 a

0.358 ± 0.024 b

FZB 37 sup

29.6 ± 13.2 b

7.5 ± 0.7 b

0.331 ± 0.068 b

FZB 38 sup

32.9 ± 7.1 a

7.4± 0.6 b

0.351 ± 0.035 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20 for control, FZB24 sup and FZB37 sup; n = 15 for FZB38 sup). Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

Of all three tested supernatants of B. subtilis (Figures 7, 8, 9, 11, 12, 13, 14), the supernatant of strain FZB38 in the test on A. fabae was found to present the best correlation between RGR and rm values (Figure 10). In the R. padi test, we observed only a weak correlation between RGR and rm values for the three strains of B. subtilis (Figures 12, 13, 14). In the same test, the control (water-treated) showed a relative higher correlation between the RGR and rm (Figure 11).

Figure 7: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with water (control), r2= 0.4504

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Figure 8: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with supernatant of Bacillus subtilis strain (FZB24), r2= 0.2019

Figure 9: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with supernatant of Bacillus subtilis strain (FZB37), r2= 0.3842

Figure 10: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with supernatant of Bacillus subtilis strain (FZB 38), r2= 0.5440

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Figure 11: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Rhopalosiphum padi feeding on Triticum aestivum treated with water (control), r2= 0.4422

Figure 12: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Rhopalosiphum padi feeding on Triticum aestivum treated with supernatant of Bacillus subtilis strain (FZB24), r2= 0.0203

Figure 13: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Rhopalosiphum padi feeding on Triticum aestivum treated with supernatant of Bacillus subtilis strain (FZB37), r2= 0.1604

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Figure 14: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Rhopalosiphum padi feeding on Triticum aestivum treated with supernatant of Bacillus subtilis strain (FZB38), r2= 0.2375

3.1.6. Influence of pre-treatment of Vicia faba foliar with spore suspensions of Bacillus subtilis strains FZB24, FZB37 and FZB38 on feeding of Aphis fabae, compared to the influence of the bacteria supernatants

As it was unclear whether the obtained induced resistance effects were specific to the supernatant, the spore suspensions of the strains FZB24, FZB37 and FZB38 of B. subtilis were directly compared to the supernatants of strains FZB24 and FZB38 of B. subtilis.

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As shown in Tables 15 and 16, the spore suspensions of the mentioned strains of B. subtilis could not reduce the growth parameters of A. fabae - the supernatants alone were able to achieve this. In supernatants of B. subtilis treatments, A. fabae and T. aestivum presented reduced adult weight, longer tDand td compared to the spore suspension treatments. The number of insect larvae born to A. fabae and T. aestivum in a time period of 2 × (td), Md, was found to be very low in supernatant treatments compared to the spore suspension treatments. The same observation was made for the values of RGR and rm (Tables 15, 16).

Table 15: Influence of spore suspensions of three strains (FZB24, FZB37, FZB38) of Bacillus subtilis applied onto the foliage of Vicia faba seedlings compared to supernatant from strains, FZB24 and FZB38 of Bacillus subtilis. The growth parameters of Aphis fabae were assessed.

Treatment

A (mg)

B (m)g

tD

(days)

RGR

Control

0,036

0.627 ± 0.048 a

6.2 ± 0.4 a

0.464 ± 0.029 a

FZB24 sps

0.039

0.634 ± 0.066 a

6.4 ± 0.5 a

0.441 ± 0.037 a

FZB37 sps

0.036

0.611 ± 0.058 a

6.1 ± 0.5 a

0.430 ± 0.039 a

FZB38 sps

0.043

0.654 ± 0.081 a

6.2 ± 0.4 a

0.439 ± 0.030 a

FZB24 sup

0.036

0.449 ± 0.122 b

6.9 ± 0.4 b

0.363 ± 0.042 b

FZB38 sup

0.033

0.416 ± 0.119 b

6.8 ± 0.4 b

0.371 ± 0.065 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20). This experiment was performed at theBA under greenhouse conditions.
A: Average mean weight of newly born insect larvae (L1); B: Weight of adult of individual insect; tD: development time, time from birth to adult moult; RGR: Relative growth rate

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Table 16: Influence of spore suspensions of three strains (FZB 24, FZB 37, FZB 38) of Bacillus subtilis applied onto the foliage of Vicia faba seedlings compared to supernatant from strains, FZB 24 and FZB 38 of Bacillus subtilis. The growth parameters of Aphis fabae were assessed.

Treatment

Md

td

(days)

rm

Control

44.6 ± 6.9 a

6.8 ± 0.4 a

0.406 ± 0.027 a

FZB 24 sps

44.0 ± 7.1 a

7.0 ± 0.5 a

0.396 ± 0.023 a

FZB 37 sps

44.1 ± 7.7 a

7.0 ± 0.5 a

0.391 ± 0.033 a

FZB 38 sps

47.1 ± 9.5 a

7.2 ± 0.4 a

0.402 ± 0.030 a

FZB 24 sup

29.15 ± 6.9 b

7.8 ± 0.4 b

0.324 ± 0.032 b

FZB 38 sup

30.7 ± 9.0 b

7.5 ± 0.6 b

0.330 ± 0.400 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test, p< 0.05; n = 20). This experiment was performed at the BBA under greenhouse conditions.
Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

In this experiment the following correlations (Figures 15, 16, 17, 18) were found between the RGR and rm values for each treatment, showing that those insects with bigger RGR also have higher rm. The correlations were verified for the control (water-treated), with more than 68 % of the test insects (Figure 15), the spore suspensions of B. subtilis strain FZB 37, more than 74 % (Figure 16) and supernatant of B. subtilis strain FZB38, approximately 42 % (Figure 18). The spore suspensions of B. subtilis strain FZB38 showed a weak correlation with an approximate estimate of only 24 % of the tested insects (Figure 17).

Figure 15: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with water (Control), r2= 0.6861

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Figure 16: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with spore suspensions of Bacillus subtilis strain (FZB37), r2= 0.7489

Figure 17: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with spore suspensions of Bacillus subtilis strain (FZB38), r2= 0.2447

Figure 18: Intrinsic rate of natural increase (rm) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with supernatant of Bacillus subtilis strain (FZB 38), r2= 0.4174

3.1.7.  Influence of pre-treatment of Vicia faba seedlings with vegetative cells of Bacillus subtilis strains FZB24, FZB37 and FZB38 on feeding of Aphis fabae

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Host plant foliage pre-treatment with vegetative cells of B. subtilis strain FZB24, FZB37 and FZB38 was unable to significantly influence the growth parameters of A. fabae feeding on V. faba (Tables 17, 18). However, following this treatment we could observe longer tD and td, as opposed to slightly diminished rm. However, this result was not statistically verified.

Table 17: Influence of vegetative cells from Bacillus subtilis strain FZB24, FZB37 and FZB38 at transition phase on Aphis fabae feeding on Vicia faba

Treatment

A (mg)

B (mg)

tD

(days)

RGR

Control

0.037

0.630 ± 0.120 a

6.0 ± 0.0 a

0.469 ± 0.032 a

FZB 24 vc

0.034

0.609 ± 0.127 a

6.2 ± 0.4 a

0.464 ± 0.055 a

FZB 37 vc

0.036

0.576 ± 0.117 b

6.1 ± 0.2 a

0.455 ± 0.039 a

FZB 38 vc

0.038

0.635 ± 0.090 a

6.1 ± 0.2 a

0.465 ± 0.029 a

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test; n = 20; p < 0.05).
A: Average mean weight of newly born insect larvae (L1); B: Weight of adult individual insect; tD: development time, time from birth to adult moult; RGR: Relative growth rate

Table 18: Effect of Vicia faba plant pre-treatment with vegetative cells from Bacillus subtilis strain FZB24, FZB37 and FZB38 at transition phase on Aphis fabae

Treatment

Md

td

(days)

rm

Control

48.6 ± 7.9 a

7.0 ± 0.0 a

0.417 ± 0.017 a

FZB 24 vc

48.8 ± 8.8 a

7.2 ± 0.4 a

0.407 ± 0.030 a

FZB 37 vc

44.9 ± 7.1 a

7.1 ± 0.2 a

0.406 ± 0.019 a

FZB 38 vc

49.0 ± 7.2 a

7.1 ± 0.2 a

0.415 ± 0.020 a

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test; n = 20 p< 0.05).
Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

3.1.8.  Direct exposure of Aphis fabae and Rhopalosiphum padi to supernatants of B. subtilis strains FZB24, FZB37 and FZB38

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Based on the test results obtained from the pre-treated plant foliage on A. fabae and R. padi, it was necessary to continue the investigations in order to establish the causes of the positive activity found in supernatant treatments against A. fabae and R. padi feeding on their respective host plants (V. faba and T. aestivum). Thus, it was decided to expose the insects directly to the supernatants to exclude any effect from direct contact. This acute toxicity test with supernatants of B. subtilis was compared to the treatments (Tables 19, 20, 21, 22), in which the plants were topically pre-treated with the supernatant of B. subtilis strain (FZB37), and the aphids thereafter caged on the selected leaves for feeding.

The acute toxicity test showed that the growth parameters of the A. fabae and R. padi (Tables 19, 20, 21, 22) were not affected, in contrast to the plant foliage pre-treatment, which showed a significant change.

The antibiosis effect observed with supernatant treatments to A. fabae and R. padi are likely to be mediated via the plant, and not caused by direct contact between the test insects and the metabolites of B. subtilis.

↓49

Table 19: Effect of direct exposure of Aphis fabae to supernatant of Bacillus subtilis FZB24, FZB37, and FZB38

Treatment

A

(mg)

B

(mg)

t D

(days)

RGR

Control

0.033

0.610 ± 0.111 a

6.2 ± 0.4 a

0.470 ± 0.048 a

FZB 24 sup

0.038

0.673 ± 0.157 a

6.3 ± 0.5 a

0.459 ± 0.062 a

FZB 37 sup

0.038

0.680 ± 0.060 a

6.7 ± 0.5 a

0.432 ± 0.036 a

FZB 38 sup

0.037

0.609 ± 0.119 a

6.4 ± 0.5 a

0.438 ± 0.049 a

FZB 37 sup (foliage treatment)

0.040

0.491 ± 0.122 b

6.6 ± 0.7 a

0.382 ± 0.071 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test; n = 10; p< 0.05).
A: Average mean weight of newly born insect larvae (L1); B: Weight of adult individual insect; tD: development time, time from birth to adult moult; RGR: Relative growth rate

Table 20: Effect of direct exposure of Aphis fabae to supernatant of Bacillus subtilis FZB24, FZB37, and FZB38

Treatment

Md

td

(days)

rm

Control

49.1 ± 13.4 a

7.2 ± 0.4 a

0.406 ± 0.039 a

FZB 24 sup

46.4 ± 10.7 a

7.4 ± 0.5 a

0.390 ± 0.038 a

FZB 37 sup

41.7 ± 10.9 a

7.7 ± 0.5 a

0.381 ± 0.035 a

FZB 38 sup

41.7 ± 12.0 a

7.4 ± 0.5 a

0.378 ± 0.044 a

FZB 37 sup (foliage treatment)

38.4 ± 18.8 a

7.4 ± 0.7 a

0.362 ± 0.073 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test; n = 10; p< 0.05).
Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

Table 21: Effect of direct exposure of Rhopalosiphum padi to the supernatant of Bacillus subtilis strains FZB24, FZB37 and FZB38 on its growth parameters

Treatment

A (mg)

B (mg)

tD

(days)

RGR

Control

0.040

0.500 ± 0.100 a

5.9 ± 0.4 a

0.436 ± 0.042 a

FZB 24 sup

0.040

0.605 ± 0.128 a

6.2 ± 0.5 a

0.440 ± 0.048 a

FZB 37 sup

0.037

0.588 ± 0.132 a

6.3 ± 0.7 a

0.439 ± 0.052 a

FZB 38 sup

0.039

0.565 ± 0.110 a

6.0 ± 0.0 a

0.443 ± 0.032 a

FZB 37 sup

(foliage treatment)

0.034

0.359 ± 0.100 b

6.0 ± 0.0 a

0.384 ± 0.054 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test; n = 10; p< 0.05).
A: Average mean weight of newly born insect larvae (L1); B: Weight of adult of individual insect; tD: development time, time from birth to adult moult ; RGR: Relative Growth Rate

↓50

Table 22: Effect of direct exposure of Rhopalosiphum padi to the supernatant of Bacillus subtilis strains FZB24, FZB37 and FZB38 on its growth parameters

Treatment

Md

td

(days)

rm

Control

37.9 ± 9.6 a

6.5 ± 1.5 a

0.382 ± 0.089 a

FZB 24 sup

40.1 ± 12.9 a

6.8 ± 0.4 a

0.405 ± 0.041 a

FZB 37 sup

40.2 ± 13.6 a

7.0 ± 0.3 a

0.393 ± 0.044 a

FZB 38 sup

40.7 ± 7.9 a

7.2 ± 0.5 a

0.391 ± 0.034 a

FZB 37 sup

(foliage treatment)

33.3 ± 6.7 a

7.5 ± 0.6 a

0.350 ± 0.03 b

Values within the same column are mean ± standard deviations and those flanked by different letters are statistically significant (Tukey’s test; n = 10; p< 0.05).
Md: Number of insect larvae born in a period of time 2 × (td); td: pre-reproduction time; rm: Intrinsic rate of natural increase

3.1.9.  Effect of culture filtrates from Bacillus subtilis strains FZB24, FZB37 and FZB38 added to artificial diet of Aphis fabae

While the acute toxicity test discussed above provided strong evidence that the tested supernatants of B. subtilis are only active against A. fabae and R. padi via the plant, further tests were conducted to confirm this role of B. subtilis metabolites when added to artificial diet and submitted to A. fabae. This test was conducted in order to ascertain what form of influence the culture filtrate would have on the feeding of A. fabae.

Culture filtrates from B. subtilis FZB24, FZB37 and FZB38 were incorporated in the artificial diet for A. fabae and the relative growth rates of the feeding insects were assessed. As shown in Table 23, the measured RGRs of the insects were not affected. It is likely that the culture filtrate of B. subtilis has no direct effect on A. fabae.

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Table 23: Incorporation of culture filtrate of Bacillus subtilis strains FZB24, FZB37, and FZB38 into artificial diet of Aphis fabae and assessment of its growth parameters

Treatment

A (mg)

B (mg)

tD

RGR

100% diet

0.035

0.461

8

0.322

0.1% of water

0.035

0.461

8

0.322

1% of water

0.036

0.464

8

0.319

0.1% FZB 24 CF

0.037

0.466

8

0.317

1% FZB 24 CF

0.037

0.464

8

0.316

0.1% FZB 37 CF

0.037

0.456

8

0.314

1% FZB 37 CF

0.038

0.456

8

0.311

0.1% FZB 38 CF

0.038

0.470

8

0.314

1% FZB 38 CF

0.037

0.456

8

0.314

A: Average mean weight of newly born insect larvae (L1); B: Adult weight of individual insect; tD: development time, time from birth to adult moult; RGR: Relative Growth Rate

Figure 19: Aphis fabae feeding on artificial diet

3.2. Physiological tests

3.2.1.  Chlorophyll measurement and aphid biomass assessment

3.2.1.1.  Leaf treatment experiments

The chlorophyll fluorescence of Vicia faba plants was measured on the fourth leaves while A. fabae were caged on the third leaves. In the case of the T. aestivum plant, the measurement was taken on the third leaf while R. padi were caged on the second leaf. It was consequently found in both cases (V. faba and T. aestivum) that the chlorophyll fluorescence was higher in supernatant pre-treated plants with aphids caged on them, but this was not statistically verified (Tables 24, 26). The corresponding fresh weight and dry weight of the A. fabae and R. padi measured were also found to be lower compared to the control (water treatment). This was statistically verified in the case of A. fabae dry weight but not for R. padi (Tables 25, 27).

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Table 24: Influence of supernatant of Bacillus subtilis strain FZB37 on the chlorophyll fluorescence of Vicia faba leaves. The measurement was obtained on the fourth leaves of the plant while on the third leaves either 4 adults of A. fabae were fixed or an empty clip cage was fixed per plant.

Number of caged insects

Chlorophyll fluorescence yield

Control

FZB37 sup

0

0.605 ± 0.038 a

0.598 ± 0.043 a

4

0.582 ± 0.040 a

0.610 ± 0.038 a

Mean values ± standard deviations (n=8) on the same line, followed by the same letters are not significantly different (Tukey’s test; p< 0.05).

Table 25: Influence of supernatant of Bacillus subtilis strain FZB37 on the fresh weight and dry weight of Aphis fabae after 6 days sucking process on Vicia faba

Test variant

Fresh weight

in (mg)

Dry weight

in (mg)

Control

11.229 ± 9.208 a

3.122 ± 2.327 a

FZB37 sup

10.655 ± 8.634 a

2.284 ± 1.773 b

Mean values ± standard deviations (n=8) in the columns followed by various letters are statistically significant. (t - test; p< 0.05).

Table 26: Influence of supernatant of Bacillus subtilis strain FZB37 on the chlorophyll fluorescence of Triticum aestivum leaves. The measurements were obtained on the tertiary leaves while on the secondary leaves either 4 adults of R. padi or an empty clip cage were caged onto each plant.

Numbers of caged insects

Chlorophyll fluorescence yield

Control

FZB37 sup

0

0.440 ± 0.031 a

0.506 ± 0.054 a

4

0.432 ± 0.078 a

0.487 ± 0.049 a

Mean values ± standard deviations (n=8) on the same line followed by different letters are statistically significant (Tukey’s test; p< 0.05).

↓53

Table 27: Influence of supernatant of Bacillus subtilis strain FZB37 on the fresh weight and dry weight of Rhopalosiphum padi after 6 days sucking process on Triticum aestivum

Test variant

Fresh weight

in (mg)

Dry weight

in (mg)

Control

4.794 ± 1.318 a

1.586 ± 0.511 a

FZB37 sup

4.160 ± 1.093 a

1.130 ± 0.561 a

Mean values ± standard deviations (n=8) in the columns followed by the same letters are not significantly different (t - test; p< 0.05).

3.2.1.2.  Seed treatment experiments

After measuring the influence of supernatants of B. subtilis FZB37 on the chlorophyll fluorescence of pre-treated V. faba and T. aestivum, on which A. fabae and R. padi were respectively fixed for feeding, it was decided to measure the effect of seed treatment with spore suspensions of B. subtilis on the host plants of A. fabae and R. padi.

Seedlings of V. faba and T. aestivum, grown in sterile soil from seeds pre-treated with spore suspension of B. subtilis showed better growth (Figures 20, 21) and chlorophyll fluorescence yields. The higher value of chlorophyll fluorescence was statistically verified for T. aestivum seedlings but not for V. faba (Tables 28, 30). The fresh weight and dry weight of aphids feeding on such plants were negatively affected compared to the control (water-treatment) (Tables 29, 31), but these values were not statistically verified.

↓54

Table 28: Influence of seed pre-treatment with spore suspensions of Bacillus subtilis FZB37, which were sown into sterile soil substrate on the chlorophyll fluorescence of Vicia faba leaves. The measurement was obtained on the fourth leaves while on the third leaves either 4 adults of Aphis fabae or an empty clip cage were fixed onto each plant.

Number of caged insects

Chlorophyll fluorescence yield

Control

FZB37 sps

0

0.481 ± 0.043 a

0.514 ± 0.042 a

4

0.464 ± 0.062 a

0.498 ± 0.048 a

Mean values ± standard deviations (n=8) on the same line followed by different letters are statistically significant (Tukey’s test; p< 0.05).

Table 29: Influence of seed pre-treatment with Bacillus subtilis FZ37 spore suspensions on the fresh weight and dry weight of Aphis fabae feeding on Vicia faba sown in sterile soil

Test variant

Fresh weight

in (mg)

Dry weight

in (mg)

Control

52.7 ± 19.6 a

14.1 ± 5.9 a

FZB37 sps

46.3 ± 16.8 a

13.4 ± 4.7 a

Mean values ± standard deviations (n=8) in the columns followed by different letters are statistically significant (Tukey's test; p< 0.05).

Table 30: Influence of seed pre-treatment with Bacillus subtilis FZB37 spore suspensions, which were sown into sterile soil substrate on the chlorophyll fluorescence of Triticum aestivum leaves. The measurement was taken on the third leaves while on the second leaves either 4 adults of Rhopalosiphum padi or an empty clip cage were fixed on each plant.

Number of caged insects

Chlorophyll fluorescence yield

Control

FZB37 sps

0

0.448 ± 0.049 a

0.456 ± 0.056a

4

0.370 ± 0.063 a

0.504 ± 0.036 b

Mean values ± standard deviations (n=8) on the same line followed by different letters are statistically significant (Tukey’s test; p< 0.05)

↓55

Table 31: Influence of the pre-treatment of seeds with Bacillus subtilis FZB37 spore suspensions, which were sown into sterile soil, on the fresh weight and dry weight of Rhopalosiphum padi feeding on summer wheat (Triticum aestivum)

Test variant

Fresh weight

in (mg)

Dry weight

in (mg)

Control

7.3 ± 3.8 a

3.2 ± 1.7 a

FZB37 sps

5.2 ± 2.8 a

2.2 ± 1.3 a

Mean values ± standard deviations (n=8) in the columns followed by different letters are statistically significant (t - test; p< 0.05).

Figure 20: Summer wheat seedlings (Triticum aestivum). Left: seed treated with water. Right: seed pre-treated with spore suspensions of Bacillus subtilis

Figure 21: Broad bean (Vicia faba) seedlings. Left: seed pre-treated with sterile water. Right: seed pre-treated with spore suspensions of Bacillus subtilis

3.2.2. Amino acids from phloem
Total Concentration of amino acids in the phloem sap of Vicia faba

↓56

The total concentration of free amino acids increased following the leaf treatments with the supernatant and culture filtrate of B. subtilis strain FZB37, as compared to the control (water-treatment) (Figure 22).

Figure 22: Total concentration of amino acids (pmol) following topical treatment with Bacillus subtilis metabolites of Vicia faba foliage, without Aphis fabae infestation.
Bars with lines are mean ± standard deviations and those followed by different letters are statistically significant (Tukey’s test; p< 0.05).
Control: plant leaves treated with water; FZB37 sup: plant leaves treated with supernatant of Bacillus subtilis strain FZB37; FZB37 CF: plant leaves treated with culture filtrate of Bacillus subtilis strain FZB37

Following A. fabae infestation, the total concentration of free amino acids in control and culture filtrate treatments increased (Figure 23), whereas in plants treated with supernatant of B. subtilis strain FZB37 no major variation was observed. For a statistical overview of this observation, Figure 22 and Figure 23 were compared.

↓57

Figure 23: Total concentration of amino acids (pmol) following topical treatment with Bacillus subtilis metabolites of Vicia faba foliage with Aphis fabae infestation
Control: Plant leaves treated with water; FZB37 sup: Plant leaves treated with supernatants of Bacillus subtilis strain FZB37; FZB37CF: plant leaves treated with culture filtrate of Bacillus subtilis strain FZB37.
Bars with lines are mean ± standard deviations and those followed by different letters are statistically significant (Tukey’s test; p < 0.05).

Figure 24: Total concentration of amino acids (pmol) in phloem sap of Vicia faba. In the same group of bars, the open bars represent cases with 0 insects caged on the plant leaves, while the shaded bars show treatments with 6 insects caged on the selected plant leaves.
Bars with lines are mean ± standard deviations and groups of bars followed by different letters are statistically significant (Tukey’s test; p < 0.05).

As indicated in Figure 24, treatment with supernatant of B. subtilis FZB37 caused no variation in the total concentration of amino acid when compared to the same group of open bars (0 insects caged on treated plant leaves) and shaded bars (6 insects caged on treated plant leaves). In contrast, there is a noticable difference in total concentration of amino acid between the two groups of bars in the control and culture filtrate treatments. This observation suggests a qualitative difference between the culture filtrate and the supernatant of B. subtilis strain FZB37. Feeding activity of A. fabae mobilizes more amino acids in the case of water and culture filtrate treated plants.

↓58

Individual amino acids from phloem sap of Vicia faba .

In Figure 25 with no insect feeding on the pre-treated plants, changes were observed for the concentration of only three individual amino acids, histidine, alanine and methionine, in the control, FZB sup 37 and FZB CF 37 treatments.

In contrast with A. fabae feeding on pre-treated plants, a marked change occurred in individual amino acids and was found to be significant for nine of them, as indicated in Figure 26. The histidine level is higher in FZB37 CF treatment than the control and FZB37 sup treatments. Glycine and threonine concentrations in FZB37 CF treatment were found to be higher than in FZB37 sup treatment, but were not different from the control (water treatment). Alanine was found to be in a higher concentration in the FZB37 sup treatment than the control but not different from that in the FZB37 CF. Tyrosine level in FZB37 sup treatment was higher than control treatment but not different from FZB37 CF treatment. Leucine and lysine were both present in a higher concentration in the FZB37 CF treatment, when compared to the control and FZB 37 sup treatments. Serine and asparagine concentrations in FZB37 sup treatment were lower when compared to the control and FZB37 CF treatments. Comparing Figure 25 and Figure 26, we can observe that the concentration of serine did not change for FZB37 sup treatment, as opposed to the control and FZB37 CF treatments, suggesting that the supernatant is capable of protecting the integrity of this amino acid, vital for insect growth in general and particularly for, in this case, A. fabae.

↓59

Figure 25: Phloem concentration of amino acids (pmol) from pre-treated and untreated Vicia faba plants, without Aphis fabae infestation. Bars with lines are means ± S.E. Groups of bars followed by different letters are statistically significant (Tukey’s test; p < 0.05).
Alanine (ala), arginine (arg), asparagine (asp), cysteine (cys), glutamine (glu), glycine (gly), histidine (his), isoleucine (ile), leucine (leu), lysine (lys), methionine (met), phenylalanine (phe), serine (ser), threonine (thr), tyrosine (tyr), valine (val)

Figure 26: Phloem concentration of amino acids (pmol) from pre-treated and untreated Vicia faba plants on which 6 adults of Aphis fabae had been caged for 6 days. Bars with lines are means ± standard deviations. Groups of bars followed by different letters are statistically significant (Tukey’s test, p < 0.05).
Alanine (ala), arginine (arg), asparagine (asp), cysteine (cys), glutamine (glu), glycine (gly), histidine (his), isoleucine (ile), leucine (leu), lysine (lys), methionine (met), phenylalanine (phe), serine (ser), threonine (thr), tyrosine (tyr), valine (val)

3.3. Reisolation of spores of B. subtilis from sterile soil substrate cultivated with Vicia faba and Triticum aestivum

As demonstrated by Thonart et al. (1994), B. subtilis can develop better if inoculated in sterile soil passing from 105 cells/g to 108 cells/g of soil. This was confirmed by the results of our experiment, in which spores of B. subtilis strain FZB 37, with which V. faba and T. aestivum seedlings had been innoculated at a concentration of 105 cfu/ml and cultivated in the soil, were reisolated and counted. In both plant systems higher colonies of organisms resembling B. subtilis were found (Table 32).

↓60

Table 32: Number of spores of Bacillus subtilis strain (FZB37) reisolated from the soil substrate and root material of Vicia faba and Triticum aestivum seedlings expressed as [108 per g dry weight of roots/ substrate]. For both plant species, Vicia faba and Triticum aestivum, 4 corresponding insects or an empty clip cage were caged on their selected leaves.
FZB37 sp: Bacillus subtilis strain FZB37 spore suspensions; titer: 105 cfu/ml

Seed

Treatment

Vicia faba

Triticum aestivum

Rhizosphere

Soil substrate

Rhizosphere

Soil substrate

FZB37 sp

(Treatment with 4 insects)

150

142

118

108

FZB37 sp

(Treatment without insect)

140

133

123

113

Distilled water (Treatment with 4 insects)

0

0

0

0

Distilled water (Treatment without insect)

0

0

0

0


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