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Effect of nutrient supplements on destruxin

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Effect of aminoacid and hydroxy acid supplements on dtx recovery from ... Control refers to M. anisopliae grown on CZ CN 3 medium without any supplements ... – PowerPoint PPT presentation

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Title: Effect of nutrient supplements on destruxin


1
Effect of nutrient supplements on
destruxin production from the culture filtrates
of high and low virulent isolates of
Metarhizium anisopliae. K.Sowjanya Sree, V.
Padmaja Department of Botany Andhra
University Visakhapatnam, India
2
  • Metarhizium anisopliae (Metch.) Sorokin
  • Hyphomycete fungus
  • Broad range arthropod pathogen
  • Causes green muscardine disease
  • in diverse insects
  • Used in biological control of
  • several insect pests
  • Produces toxic secondary metabolites
  • like destruxins and
  • cytochalasins
  • Registered mycoinsecticides Bioblast- USA

  • Green Muscle- South Africa

3
  • The insecticidal secondary metabolite Destruxin
  • First isolated from Oospora destructor, now
    renamed as Metarhizium anisopliae (Metch.)
  • Insecticidal mycotoxin
  • Destruxin General structure
  • Cyclic hexadepsipeptides
  • 1 a- hydroxy acid
  • 5 aminoacid residues
  • Classified based on R group
  • 35 analogues reported
  • Dtx A, B and E are predominant

4
  • Insecticidal activity of destruxins- current
    status
  • Insecticidal activity Dtx A and E (predominant)
  • Specific dose dependent reversible inhibitor of
    vacuolar type ATPase Dtx B
  • Reduces cyclin D1 levels effecting cell cycle
    Dtx E
  • Mediates specific down regulation of AMPs (Anti
    Microbial Peptides) by means of
  • targeting the insects innate immune
    signalling pathway Dtx A
  • Induces oxidative stress in the treated larvae
    changing the profile of the enzymatic
  • and non- enzymatic antioxidants Crude dtx
  • Known target organs in the lepidopteran larval
    body include- Midgut,
  • Malphigian tubules, Haemocytes, Salivary
    glands Dtx A, Crude dtx

5
  • Objectives of the current study
  • To optimize the recovery of dtx A and E from the
    culture filtrates of
  • M. anisopliae grown on Czapek Dox (CZ) medium
    with
  • varying C / N ratios.
  • To enhance the production of dtx A and E in the
    culture filtrates of M. anisopliae
  • by supplementing CZ CN 3 medium with different
    aminoacid and
  • hydroxy acid precursors.
  • To study the biochemical profiles of the mycelium
    of M. anisopliae grown on
  • different nutrient supplements

6
In vitro destruxin production and extraction
50 ml of CZ minimal medium inoculate with 1
ml M. anisopliae spore suspension (2x107)

incubate at
25C and 200 rpm for
7 days in a rotary shaker culture filtered
through layers of Whatmans no.1 filter
paper Extract filtrate twice for every 12 hours
with DCM EtOAc (11)
solvent
evaporation Crude extract dissolved in 1 ml
methanol (HPLC grade)
7
  • Detection of destruxins
  • TLC with flourescence indicators
  • Reverse phase HPLC with UV detection at 206- 214
    nm
  • Quantification of destruxins
  • HPLC Peak area measurements in comparison with
    the standard.

8
Strains of M. anisopliae used in the
current investigation High Virulent
strain M-19 from Agricultural
research services collection of entomopathogenic
fungi (ARSEF), ITHACA
collection (ARSEF- 1080)
and Low
virulent strain M-10 from
Empresa Brasileira de Pesquisa Agropecuária
(EMBRAPA), Brazil (CG- 47).
9
Crude destruxin
Reverse phase high performance liquid
chromatographic (RPHPLC) profile of crude
destruxin from M-19 strain of M. anisopliae. The
two major forms of dtx dtx A and dtx E detected
in the crude dtx recovered from the cultures
after 7 days post inoculation.
10
  • Effect of nutrient supplements on destruxin
    production
  • Medium composition
  • C / N ratio
  • supplements like aminoacids and hydroxy acid
  • The other conditions were kept constant
  • pH- 7.0
  • Temperature- 25 C
  • Rpm - 200

11
  • C / N Ratio
  • The Different C / N ratios used in the this
    study were
  • Glucose (g / L)
    Peptone (g / L)
  • CN 1 30
    0
  • CN 2 0
    30
  • CN 3 25
    5
  • CN 4 20
    10
  • CN 5 15
    15
  • CN 6 10
    20
  • CN 7 5
    25

12
  • Supplements to the CD CN 3 medium
  • Aminoacids
  • b- Alanine (ß- Ala)
  • Methionine (Met)
  • Proline (Pro)
  • Isoleucine (Ile)
  • Valine (Val)
  • Hydroxy acid
  • Sodium acetate (Na acetate)

13
Effect of different C / N ratios on the pellet
size and dtx recovery from
low virulent M-10 strain of M. anisopliae
14
Effect of different C / N ratios on the pellet
size and dtx recovery from
high virulent M-19 strain of M. anisopliae
15
Effect of aminoacid and hydroxy acid supplements
on dtx recovery from
high virulent M-19 strain grown on CZ CN 3
16
  • Biochemical profile of high virulent M-19 strain
    of M. anisopliae
  • grown on CZ CN 3 medium with aminoacid and
    hydroxy acid supplements
  • in terms of
  • Acid phosphatase
  • Alkaline phosphatase
  • Esterase

17
Enzyme activity in the pellets of M-19 strain of
M. anisopliae grown on
supplemented CZ CN 3 medium
18
Isozyme profile of Acid phosphatase in M-19
strain of M. anisopliae
grown on supplemented CZ CN 3 medium
Control refers to M. anisopliae grown on CZ CN 3
medium without any supplements
19
Isozyme profile of Alkaline phosphatase in M-19
strain of M. anisopliae
grown on supplemented CZ CN 3 medium
Control refers to M. anisopliae grown on CZ CN 3
medium without any supplements
20
Isozyme profile of Esterase in M-19 strain of M.
anisopliae grown on
supplemented CZ CN 3 medium
Control refers to M. anisopliae grown on CZ CN 3
medium without any supplements
21
  • Important observations
  • CZ medium with CN 3 composition resulted in
    optimum production of dtx A and dtx E from
    both M-19 and M-10 strains of M. anisopliae.
    However, maximum quantities were recovered from
    the high virulent M-19 strain (dtx A- 10.8 mg/ L
    and dtx E- 8.28 mg/ L).
  • Dtx recovery directly correlated with the fungal
    pellet size in both M-19 and M-10 strains.
  • High virulent M-19 strain grown on supplemented
    CZ CN 3 medium produced higher amounts of dtx A
    and E. The maximum was recorded with ß- Alanine
    (two fold increase, dtx A- 18.9 mg/ L and dtx E-
    15.3 mg/ L) followed by Isoleucine and Proline.
  • Maximum increase in the activity of Acid
    phosphatase, Alkaline phosphatase and esterase
    enzymes in M-19 strain of M. anisopliae grown on
    CZ CN 3 medium supplemented with ß- Alanine,
    Isoleucine and Proline. This trend was also
    observed in the isozyme profile of these enzymes.

22
Destruxin - a virulence factor ?
Correlation between in vitro destruxin production
and fungal virulence- ( M-19, a high
virulent strain producing higher amount of
destruxin than the low virulent M-10 strain)

23
  • Insecticidal formulation of destruxin- Current
    status
  • Research directed towards developing destruxin/
    destruxin in combination with conidia
  • of Metarhizium anisopliae as a bioinsecticide
  • Crude destruxin is also recommended for
    developing a cost effective insecticidal
    formulation
  • Laverlam SA company located in Cali, Colombia
    manufactures a registered product by
  • the trade name DESTRUXIN
  • This product has passed tier 1 test for
    microbial pest control agents of the
  • US EPA
  • Marketed in South American countries like
    Uruguay, Venezuela, Bolivia,
  • Ecuador, Peru, Costa Rica, Dominican Republic,
    Honduras and Panama
  • Wettable powder formulation containing conidia,
    mycilia and destruxin of
  • M. anisopliae,fermentation solids and
    stabilizers

24
  • Conclusions
  • Destruxins are of great interest due to their
    significant insecticidal activity
  • Dtx recovery is maximum in the CZ CN 3 medium
    supplemented with ß- Alanine
  • followed by Isoleucine and Proline.
  • This is possibly because of their key role in
    the cyclization process of the synthesis
  • this cyclodepsipeptide.
  • Future outlook
  • Potential pesticidal properties of destruxin to
    be exploited for development of an
  • eco friendly mycopesticide for use in the
    integrated pest management programmes.

25
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