September 22, 2015

Annie Chen
Bioprospecting

A Mixed Culture of Endophytic

Fungi Increases Production
of Antifungal Polyketides
By Fernanda O. Chagas, Lus G. Dias, and Mnica T. Pupo
University of So Paulo, Ribeiro Preto, So Paulo, Brazil

Introduction
Secondary

metabolites
produced by endophytes
may be beneficial to host
plants
Stimulates growth
Resistance to biotic/abiotic
factors

Studies

have been done

on the relationship
between single
endophytes and their host
plants, but not on the
effect of the relationship
between co-habiting
endophytes on their host.

Endophytes

do not
express certain genes
under artificial lab
conditions
Most previous studies tried
to obtain natural products
from axenic monocultures
Fix: grow fungi in mixed
cultures

Purpose:

examine the
chemical potential of
mixed cultivation of five
different endophytes
isolated from the same
host plant

Zhu F, Lin Y (2006) Marinamide, a novel alkaloid and its methyl ester produced by the application of mixed fermentation technique to two mangrove
endophytic fungi from the South China Sea. Chin Sci Bull 51:14261430
Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19:792798

Materials and Methods

Smallanthus sonchifolius
(Asteraceae)

Commonly known as the

yacn

Perennial daisy grown in

South America for its
potato-like tubers

The leaves also have

medicinal value (gives
tisanes prebiotic and
antioxidant properties)

Can grow up to 2m in height

and produces small yellow
flowers

Close relative to the

sunflower and Jerusalem
artichoke

Stock fungi were maintained

on mineral oil, agar plugs in
sterile water, and silica gel at
4 degrees Celsius

http://cipotato.org/roots-and-tubers/yacon

Single and Mixed Liquid Culture

Pre-cultures grown
on Potato Dextrose
Agar (PDA)

A liquid-liquid
partition was also
used (ethanolic
solution and ethyl
acetate)

Fungal Antagonism Assay in Semi-Solid

Medium

Pure cultures were directly

extracted

Mixed cultures were divided

into 3 extractions
Fungus #1 (1)
Zone of inhibition (2)
Fungus #2 (3)

Competition cultures were prepared with

one plug of each endophyte

Examination

1 mL/min

Reversed-phase C18
analytical column attached to
guard column using gradient
solving system with aqueous
acetonitrile for 30 minutes

20 L of each sample
analyzed at 1 mg/mL

Diode-array detection
continuously measures UV
absorption at multiple
wavelengths

Five polyketides analyzed:

Stemphyperylenol
Altertoxin I
Alterperylenol
Alternariol
Alternariol monomethyl ether

Antagonism Due to Diffusible Compounds

Control: A. tenuissima
grown without previous
inoculation

Two replicas of each plate

Antagonism Due to Volatile Compounds

Control: some lids were

replaced with clean ones

Antifungal Activity Assay

Positive control: actidione

Negative control: no
compound

Spore suspension: 10
mL PDB + PDA dish of
21-day-old N.
sphaerica

Cell viability indicator:

resazurin

Phytotoxicity Assay

Results & Discussion

Fungal

strains were chosen based

on the cytotoxicity of their pure
crude extracts against tumor cells

During

initial screening, only A.

tenuissima and N. sphaerica showed
significant chemical difference
between the mixed and pure
cultures.
Polyketide stemphyperylenol was
exclusive to mixed cultures
Although all five polyketides were
detected in small amounts in the pure
culture of A. tenuissima?

No

new compounds found, but

experimental data agrees with
published data

Mixed

cultures grew slower than

pure cultures meant inhibition
mechanisms at work
Combination of polyketides may be
responsible for growth inhibition

Results & Discussion

Pink = N. sphaerica pure culture extract

Blue = A. tenuissima pure culture extract

Black = mixed culture extract

Results & Discussion

Semi-solid

media (PDA)

N. sphaerica and A. tenuissima had the most significant effect

on each other; they had the largest zone of inhibition
After

mass-producing the extracts, the five polyketides were detected

Hypothesis:

Diffusible

polyketides may inhibit growth of N. sphaerica

compounds

Produced by A. tenuissima: yes

Produced by N. sphaerica: no
Volatile

compounds

No change detected compared to positive control

Thus, inhibition is only due to diffusible compounds through
medium
So

what?

Direct interaction in liquid culture was important for production

of diffusible compounds
A. tenuissima may be producing polyketides to prevent N.
sphaerica from taking over the host

Results & Discussion

Antifungal

assay

Stemphyperylenol blocked N. sphaericas growth better than the

known antifungal actidione (positive control) at 200 M
concentration
Phytotoxicity

assay

Stemphyperylenol did not damage leaves after 7 days, even at

concentrations as high as 2 mM
Significant compared to positive control which damaged leaves in
24 hours

Conclusion
Stemphyperylenol

is known to be present in A.
tenuissima as well as other fungal species
Belongs to perylene quinone class
Other

compounds in same class are phytotoxic

Polyketides

may play a part in

maintaining endophyte-host plant
symbiosis

Further Research
Test

all five polyketides together

Isolate

fungi from leaves