Bacillus subtilis und seine Stoffwechselprodukte als Agenzien zur Resistenzinduktion gegen Blattläuse auf Ackerbohne (Vicia faba) und Sommerweizen (Triticum aestivum).

Dissertation

Zur Erlangerung des akademischen Grades
Doctor Agriculturarum
(Dr. rer. agr.)

Eingereicht an der
Landwirtschaftlich-Gärtnerischen Fakultät
der Humboldt-Universität zu Berlin

von

(M.Sc. Agric., M.Sc. Development Studies, Vinaman Yao )

(Geb. am 17.12.
1961, in Treichville, Côte d’Ivoire)

Präsident der Humboldt-Universität zu Berlin

Prof. Dr. Jürgen Mlynek

Dekan: der Landwirtschaftlich-Gärtnerischen Fakultät
Prof. Dr. Uwe Jens Nagel

Gutachter:
1. Helmuth Bochow
2. Christoph Reichmuth
3. Hans -Holger Liste
4. Geoffrey Zehnder

Tag der mündlichen Prüfung: 20.01.06

Zusammenfassung

Bacillus subtilis und seine Stoffwechselprodukte als Agenzien zur Resistenzinduktion gegen Blattläuse auf Ackerbohne ( Vicia faba ) und Sommerweizen ( Triticum aestivum ) - Dissertation Humboldt-Universität Berlin; 32 Tab., 26 Abb.

Bacillus subtilis Stämme vom FZB - FZB24, FZB37 und FZB38 des FZB Biotechnik, Berlin - und ihre Stoffwechselprodukte wurden als Agenzien für eine Resistenzinduktion gegen Schädiger in drei Wirtspflanzen-Erreger-Systemen, Vicia faba - Uromyces appendiculatus, Vicia faba - Aphis fabae und Triticum aestivum - Rhopalosiphum padi untersucht. Prä-inokulativ wurden Blätter und Saatgut der Pflanzen mit den Testsubstanzen behandelt. Zur Testung gelangten Bakterien-Kulturfiltrat (KF), KF-Zenrifugationsüberstände, die vegetativen Zellen und Sporen der B. subtilis Stämme.

Bei lokaler und systemischer Pflanzenbehandlung riefen Bakterien-Kulturfiltrate und KF-Überstände eine Entwicklungshemmung der Uredosporen des Rostpilzes U. appendiculatus hervor.

Die Entwicklung der Blattläuse A. fabae und R. padi auf den Wirtspflanzen wurde mit Hilfe der Lebenstafel-Methode bewertet, wobei die Entwicklungszeit der Tiere (t_D), die Prä-Reproduktionszeit (t_d), die Relative Wachstumsrate (RGR) und die natürliche Wachstumsrate (r_m) ermittelt wurden.

Nach lokaler Applikation der KF-Überstände von B. subtilis auf die Wirtspflanzenblätter konnte eine erhebliche Breite antibiotischer (entwicklungshemmender) Wirkungen auf A. fabae und R. padi beobachtet werden. Die Aphiden benötigten längere Entwicklungs- und Prä-Reproduktionszeiten und gegensätzlich konnte eine geringere Relative (RGR) und natürliche Wachstumsrate (r_m) beobachtet werden.

Diese in den Versuchen festgestellten antibiotischen Wirkungen wurden offensichtlich durch die Pflanze vermittelt. Direkte toxische Effekte der bakteriellen Stoffwechselprodukte auf die Testtiere A. fabae und R. padi waren nicht erkennbar.

Eine Untersuchung der freien Aminosäuren im Phloemsaft von Vicia faba zeigte, daß sichnach Befall von Aphis fabae speziell bei den zuvor mit bakteriellem Kulturfiltrat und KF-Überstand behandelten Pflanzen im Vergleich zu der nur mit Wasser behandelten Kontrolle, die Konzentration von neun Aminosäuren änderte. Bei den mit KF-Überstand behandelten Pflanzen blieb auffallend die Konzentration der Aminosäure Serin unverändert, was als ein Hauptgrund für den bei diesen Pflanzen beobachteten antibiotischen Effekt auf die getesteten Aphiden interpretiert wird.

Neben der Induktion antibiotischer Wirkungen ließ sich nach der Pflanzenbehandlung mit Bacillus subtilis und seinen Stoffwechselprodukten eine höhere Chlorophyllfluoreszenz und ein besseres Wachstum der Pflanzen feststellen.

Eine Induktion von Resistenz gegenüber Aphis fabae und Rhopalosiphum padi wird durch Vorbehandlung der Wirtspflanzen mit dem KF-Überstand der

B. subtilis Stämme FZB24, FZB37 und FZB38 als nutzbar gesehen.

Weiterführende Untersuchungen sollten sich auf das Vorkommen sekundärer Metaboliten orientieren, besonders auf Proteinase inhibierender Proteine, im Phloemsaft mit KF-Überständen von Bacillus subtilis behandelter Pflanzen.

Eigene Schlagworte: Induzierte Resistenz, Ackerbohne, Sommerweizen, Bacillus subtilis , Aphis fabae , Rhopalosiphum padi

Abstract

Bacillus subtilis and its metabolites as induced resistance agent against aphids feeding on broad bean ( Vicia faba ) and summer wheat ( Triticum aestivum )

Strains of Bacillus subtilis FZB (FZB24, FZB37 and FZB38 from FZB Biotechnik Berlin) and its metabolites were investigated for their role in induced resistance in three host–parasite systems, i.e. Vicia faba - Uromyces appendiculatus, Vicia faba - Aphis fabae and Triticum aestivum - Rhopalosiphum padi, following plant foliar and seed treatment. The culture filtrate, supernatant, vegetative cells and spore suspensions of the different strains of Bacillus subtilis were examined.

In topical and systemic plant treatment, the culture filtrate and supernatant of Bacillus subtilis was shown to inhibit the development of urediospores produced by Uromyces appendiculatus. The performance of Aphis fabae and Rhopalosiphum padi was evaluated using life table tests where the aphids' development time (t_D), pre-reproduction time (t_d), relative growth rate (RGR) and intrinsic rate of natural increase (r_m) were assessed. A wide range of antibiosis effects in Aphis fabae and Rhopalosiphum padi was observed when the supernatant of Bacillus subtilis was used as foliar topical treatment. The tested aphids presented longer development and pre-reproduction time; conversely a lower relative growth rate and intrinsic rate of natural increase was observed.

The antibiosis effect observed in the study was likely mediated via the plant. No direct toxicity effect of Bacillus subtilis metabolites on Aphis fabae and Rhopalosiphum padi was observed.

The investigation of the free amino acids of the phloem sap of V. faba plants, showed that, following A. fabae infestation, the concentration of nine individual amino acids had changed in the supernatant and culture filtrate of B. subtilis treated plants compared to the control (water treatment). The concentration of the amino acid serine remained unchanged in the supernatant induced plants in this study, which was interpreted as the major reason for the observed antibiosis effect on the tested aphids.

Besides the antibiosis, higher chlorophyll fluorescence and enhanced plant growth were evident as a result of plant treatment with Bacillus subtilis and its metabolites.

Induced resistance to both A. fabae and R. padi is suggested when using supernatants (FZB24, FZB37 and FZB38) of B. subtilis in topical treatment.

Keywords: Induced resistance, Bacillus subtilis, Aphis fabae, Rhopalosiphum padi

Table of contents

• 1. Introduction
• 2.  Material and methods
  • 2.1.  Soil substrate
  • 2.2. Plant material
    • 2.2.1.  Broad bean (Vicia faba) cv. ‘Hangdown’
    • 2.2.2. Summer wheat (Triticum aestivum) cv. ‘Nandu’
  • 2.3. Test organism breeding conditions
    • 2.3.1.  Fungi
    • 2.3.2.  A. fabae and R. padi breeding conditions
  • 2.4. Clip cages used in aphid tests
  • 2.5.  Bacillus subtilis and its metabolites
    • 2.5.1.  Biology and morphology of Bacillus subtilis (Ehrenberg) Cohn
    • 2.5.2.  Bacillus subtilis metabolite productions
      • 2.5.2.1.  Spore suspensions
      • 2.5.2.2. Culture filtrate
      • 2.5.2.3. Supernatant
      • 2.5.2.4. Vegetative cells
  • 2.6. Utilisation of the obtained materials in the experiments
    • 2.6.1.  Seed pre-treatment with spore suspensions prior to sowing
      • 2.6.1.1.  Treatment procedure
      • 2.6.1.2. Reisolation of Bacillus subtilis from the roots
    • 2.6.2. Application of spore suspensions and vegetative cells on the leaves
    • 2.6.3. Topical treatment of the plant leaves using culture filtrate and the supernatant
    • 2.6.4. Systemic treatment of the plant leaves using culture filtrate and the supernatant
    • 2.6.5. Application of supernatants of Bacillus subtilis in acute toxicity test
  • 2.7. Rearing Aphis fabae on sterile synthetic diet
    • 2.7.1.  Synthetic diet preparation procedure
    • 2.7.2. Feeding apparatus used in sterile feeding test
  • 2.8. Artificial infestation of Vicia faba with Uromyces appendiculatus
  • 2.9. Life table tests for Aphis fabae and Rhopalosiphum padi
    • 2.9.1.  The estimation of Relative Growth Rates of Aphis fabae and Rhopalosiphum padi
    • 2.9.2. The e estimation of the intrinsic rates of natural increase (rm) of Aphis fabae and Rhopalosiphum padi
  • 2.10. Physiological tests
    • 2.10.1.  Chlorophyll fluorescence measurement
    • 2.10.2. Amino acids investigation
      • 2.10.2.1.  Sample collection
      • 2.10.2.2. Amino acids extraction and analysis
  • 2.11. Statistical analysis
• 3. Results
  • 3.1.  Influence of Bacillus subtilis metabolites on Uromyces appendiculatus, Aphis fabae and Rhopalosiphum padi
    • 3.1.1.  Uromyces appendiculatus
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 3.1.8.  Direct exposure of Aphis fabae and Rhopalosiphum padi to supernatants of B. subtilis strains FZB24, FZB37 and FZB38
    • 3.1.9.  Effect of culture filtrates from Bacillus subtilis strains FZB24, FZB37 and FZB38 added to artificial diet of Aphis fabae
  • 3.2. Physiological tests
    • 3.2.1.  Chlorophyll measurement and aphid biomass assessment
      • 3.2.1.1.  Leaf treatment experiments
      • 3.2.1.2.  Seed treatment experiments
    • 3.2.2. Amino acids from phloem Total Concentration of amino acids in the phloem sap of Vicia faba
  • 3.3. Reisolation of spores of B. subtilis from sterile soil substrate cultivated with Vicia faba and Triticum aestivum
• 4. Discussion
  • 4.1.  The patterns of resistance in different host—parasite systems
  • 4.2. The feeding habits of the aphids
  • 4.3. The role of amino acids in aphid nutrition
  • 4.4. Chlorophyll fluorescence measurement and aphids’ fresh weight and dry weight assessment
  • 4.5. Unspecific character of the inducer
  • 4.6. The future of induced resistance in aphids’ control
• 5. Conclusion
• 6. Future perspectives
• Abbreviations
• Literature
• Acknowledgements
• Declaration

Tables

• Table 1: Composition of the Landy-Medium (LM) for 1 liter of solution
• Table 2: Characteristics of culture filtrate of strain FZB24 of Bacillus subtilis in Landy-Medium (Beckmann, 1995 and FZB Biotechnik GmbH, 1995)
• Table 3: Composition of synthetic diet (mg/ 100 ml of diet)
• Table 4: Effect of Vicia faba seedlings’pre-treatment with Bacillus subtilis metabolites on the number of rust pustules produced by Uromyces appendiculatus
• 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
• 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
• 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
• 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
• 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
• 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
• Table 11: Influence of supernatants from Bacillus subtilis FZB24, FZB37 and FZB38 on growth parameters of Aphis fabae feeding on Vicia faba
• Table 12: Influence of supernatants from Bacillus subtilis FZB24, FZB37 and FZB38 on Aphis fabae feeding on Vicia faba
• Table 13: Influence of supernatants from Bacillus subtilis FZB24, FZB37 and FZB38 on Rhopalosiphum padi feeding on Triticum aestivum
• Table 14: Influence of supernatants from Bacillus subtilis FZB24, FZB37 and FZB38 on Rhopalosiphum padi feeding on Triticum aestivum
• 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.
• 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.
• Table 17: Influence of vegetative cells from Bacillus subtilis strain FZB24, FZB37 and FZB38 at transition phase on Aphis fabae feeding on Vicia faba
• 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
• Table 19: Effect of direct exposure of Aphis fabae to supernatant of Bacillus subtilis FZB24, FZB37, and FZB38
• Table 20: Effect of direct exposure of Aphis fabae to supernatant of Bacillus subtilis FZB24, FZB37, and FZB38
• Table 21: Effect of direct exposure of Rhopalosiphum padi to the supernatant of Bacillus subtilis strains FZB24, FZB37 and FZB38 on its growth parameters
• Table 22: Effect of direct exposure of Rhopalosiphum padi to the supernatant of Bacillus subtilis strains FZB24, FZB37 and FZB38 on its growth parameters
• 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
• 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.
• 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
• 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.
• 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
• 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.
• 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
• 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.
• 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)
• 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

Images

• Figure 1:Clip cages fixed on test plant leaflets; right and left on Vicia fabae and Triticum aestivum leaflet, respectively
• Figure 2: Diagram of the production of Bacillus subtilis and its metabolites. CF: Culture Filtrate; sup: supernatant; vc: vegetative cells; sp: spore suspensions
• Figure 3: Systemic induced treatment of a Vicia faba plant.
• 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.
• Figure 5: Chlorophyll fluorescence measurement of Triticum aestivum leaf, using a fluorimeter PAM-2000
• 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
• Figure 7: Intrinsic rate of natural increase (r_m) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with water (control), r²= 0.4504
• Figure 8: Intrinsic rate of natural increase (r_m) 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 (r_m) 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 (r_m) 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
• Figure 11: Intrinsic rate of natural increase (r_m) 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 (r_m) 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 (r_m) 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
• Figure 14: Intrinsic rate of natural increase (r_m) 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
• Figure 15: Intrinsic rate of natural increase (r_m) in relation to relative growth rate (RGR) for Aphis fabae feeding on Vicia faba treated with water (Control), r²= 0.6861
• Figure 16: Intrinsic rate of natural increase (r_m) 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 (r_m) 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 (r_m) 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
• Figure 19: Aphis fabae feeding on artificial diet
• 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
• 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
• 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).
• 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)