World J Microbiol Biotechnol (2008) 24:79–87
DOI 10.1007/s11274-007-9442-3
ORIGINAL PAPER
Optimization of process parameters for improved production
of bioactive metabolite by a novel endophytic fungus Fusarium sp.
DF2 isolated from Taxus wallichiana of North East India
Dip K. Gogoi Æ Hari P. Deka Boruah Æ
Ratul Saikia Æ Tarun C. Bora
Received: 1 November 2006 / Accepted: 5 May 2007 / Published online: 7 June 2007

Springer Science+Business Media B.V. 2007
Abstract An endophytic fungus having antifungal and
antibacterial properties was isolated from Taxus wallichi-
ana of Arunachal Pradesh, India. On the basis of mor-
phological and molecular characteristics, the fungus was
identified as Fusarium sp. and designated as DF2. The
fungus was optimized for growth and maximum production
of the antimicrobial agent. Media with 5% leaf extract
(w/v) supplemented with 0.1% dextrose as carbon and
yeast extract as nitrogen source favored the growth with
temperature optimum 25 ± 2C and pH 6. Incubation
period of 10 days was observed optimum for growth and
production of antimicrobial agent. Phenylalanine and
dextrose enriched basal medium promoted the antimicro-
bial agent production, whereas methionine amended in
combination with glucose promoted higher biomass
accumulation. The TLC purified active compound with UV
k-max 270 nm in ethyl acetate has got the lowest minimum
inhibitory concentration (MIC) against Bacillus subtilis,
Staphylococcus aureus and Escherichia coli and highest
against Pseudomonas aeruginosa.
Keywords Antimicrobial agent
Endophytes
Fusarium
sp.
MIC
Abbreviations
PDA Potato dextrose agar
NA Nutrient agar
MHA Muellar hinton agar
MTCC Microbial type culture collection
D. K. Gogoi
H. P. Deka Boruah (&)

R. Saikia
T. C. Bora
Biotechnology Division, Regional Research Laboratory (CSIR),
Jorhat 785006 Assam, India
e-mail: hpdekaboruah@yahoo.com; dekaboruah@yahoo.com
ATCC American type culture collection
IMTECH Institute of microbial technology
ICRISAT International crop research institute for the
semi arid tropics
MIC Minimum inhibitory concentration
MFC Minimum fungicidal concentration
qp Specific rate of product production
NCBI National centre for biotechnology information
Introduction
Endophytes are microorganisms that reside inside the liv-
ing tissue of the host plant asymptomatically (Glienke-
Blanco et al. 2002) exhibiting relationships ranging from
symbiotic to slightly pathogenic (Strobel and Bryn 2003).
Investigation on endophytic microorganisms have been
recently intensified due to the potentialities of endophytic
microorganisms in production of bioactive metabolite,
immunosuppressant, anticancer compound and biocontrol
agent (Lu et al. 2000; Pereira et al. 1993; Pereira et al.
1999; Regina et al. 2002; Selvin et al. 2004; Stinson et al.
2003; Strobel and Bryn 2003; Strobel et al. 2004; Wang
et al. 2000; Wang et al. 2002)
Hundreds of endophytic fungi isolated from traditional
Chinese pharmaceutical plants including Taxus mairei,
showed promising antitumour and antifungal activity
(Wang et al. 2002). It has also been reported that
endophytic fungi Taxomyces andreanae (Stierle et al.
1993), Pestalotiopsis microspore (Strobel et al. 1996) and
Tubercularia sp. (TF-5) (Wang et al. 2000) have got ability
to produce Taxol other than Taxus sp. In spite of their wide
application in synthesizing volatile metabolites including
cyanide and cyano-like compound, we are yet to explore
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and exploit their vast potentialities in both agriculture and
pharmaceutical sectors (Mc Afee et al. 1999).
Various factors like temperature, pH, incubation period,
salinity, carbon and nitrogen sources and amino acid plays
a major role on production of antimicrobial agent
(Adinarayana et al. 2003; Basak and Mazumdar 1975;
Digrak et al. 2001; Griffith and Saker 2003; Lethimaki
et al. 1997; Moita et al. 2005, Noaman et al. 2004; Sailer
et al. 1993; Vahidi et al. 2004).
Conservation International has identified the entire
North East (NE) India under the Indo Burma mega-
biodiversity hotzone (Mayrs et al. 2000). Although there is
sporadic reports on endophytic fungus growing on Taxus
sp. and some other gymnosperms (Bala et al. 1999; Saikia
et al. 2005) in NE India, yet its rich biodiversity needs to
be explore for the benefit of the society. Considering the
increasing demand of pharmaceutical sectors for novel
drug and drug intermediate to control dreaded diseases like
HIV, cancer and dominant infectious diseases, it was our
endeavor to characterize the antimicrobial property of an
endophytic fungus isolated from high altitude gymno-
sperms Taxus wallichiana, mostly known for its anticancer
property. In this investigation an effort has been made to
optimize the culture conditions and process parameters of a
newly isolated endophytic fungi Fusarium sp. DF2 and
facilitate improved production of the biologically active
compound.
Materials and Methods
Isolation & Identification of the endophyte
The endopytic fungus was isolated from the inner bark of
T. wallichiana obtained from, North East (NE) India, Sub-
Himalayan region, situated in 12000 ft above sea level.
Isolation was carried out by a little modification of the
method mentioned by Wang et al (2000) and further
maintained on potato dextrose agar (PDA) slant. For iso-
lation, the inner bark was cut into small pieces (~0.5 ·
0.5 · 0.5 cm) and then treated with 70% (v/v) ethanol
followed by 3% sodium hypochlorite (NaOCl) and the
outer bark was removed with a sterilized sharp blade.
Treated barks were placed on the surface of PDA medium
and incubated at 25C in stationary condition for 2 weeks
at 25C. Hyphal tips of the fungus, protruding from the
inner bark fragments on the plate were further purified.
Fungal identification was based on the morphology of
the fungal culture, characteristics of the spores (Ainsworth
et al. 1973, Hawksworth et al. 1983) and 18s rDNA
homology. The sequencing for 18s rRNA gene homology
was carried out at MTCC and gene bank, IMTECH,
Chandigarh, India.
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In-vitro antibiosis
In-vitro antimicrobial bioassay of the isolated fungus was
carried out by conventional agar well diffusion (Schillinger
and Lucke 1989) and dual culture technique as described by
Kang et al (2004). Pathogenic bacterial and fungal suspen-
sion were prepared in Ringer’s solution and adjusted to a final
inoculums size of 3 · 105 colony forming units (cfu/ml). In
agar well diffusion method pathogenic microbial suspensions
were spread over nutrient agar (NA), muellar hinton agar
(MHA) and PDA medium and wells of 6 mm diameter were
prepared. Equal volumes of culture filtrate were loaded into
the wells and clear inhibition zones were measured after 48 h
of incubation. In case of dual culture technique, the target
fungal pathogen and the strain DF2 were inoculated in
opposite end of the Petri dishes and zones of inhibitions were
recorded after 5 days of incubation at 30C.
Test organisms
Out of seven bacterial test pathogens, four were Gram
positive- Bacillus subtilis (NCIM2063), Staphylococcus
aureus (MTCC 737), Mycobacterium smegmatis
(ATCC11758), Mycobacterium phlei (ATCC19420) and
three Gram negative bacteria Escherichia coli (MTCC739),
Klebsiella pneumoniae. (MTCC109) and Pseudomonas
aeruginosa (MTCC741). The fungal human pathogen
Candida albicans (MTCC 3017) including the bacterial
pathogens were obtained through the courtesy of MTCC
and Gene Bank, IMTECH, Chandigarh, India.
The plant pathogens Fusarium oxysporum sp. Raphani
Schlect, and Fusarium semitectum Berkeley and Ravenel,
were obtained from ICRISAT, Hyderabad, India.
Determination of MIC and MFC
Inoculum of equal size for both bacterial and fungal test
organisms were added to 5 ml of nutrient broth (NB) and
potato dextrose broth (PDB) media respectively on differ-
ent tubes. Serial dilutions of the active compound were
added at the same time. MICs of the bacterial test patho-
gens were determined after 48 h of incubation by removing
10 ll of the contents from each tube and spreading them
onto NA plates. After 24 h of incubation at 35C growth of
any colony was observed. MFCs were determined in
molten M2 agar base (M2) by following the same proce-
dure and only the incubation period was extended upto
48 h at 28C.
Extraction and fractionation of compound
For extraction of the bioactive compound 20 ml of 48 h old
culture of Fusarium sp. DF2 prepared by incubating active
World J Microbiol Biotechnol (2008) 24:79–87
mycelial mat in 50 ml PD broth in 250 ml Erlenmeyer
flasks at 25C in a rotary shaker (150 rpm). Then 20 ml of
enrich culture were transferred as seed into each of total of
50 nos. of 500 ml Erlenmeyer flasks containing 200 ml
PDB medium each and incubated for 10 days at 25C in
stationary condition. To isolate the bioactive molecule, the
cell mass was separated by centrifugation at 7796 · g (SS-
34 rotor, RC5-B Plus Centrifuge, Sorval) for 10 min and
then the supernatant was extracted thrice with equal vol-
ume of ethyl acetate (1:1). The supernatant was treated
with Na2SO4, evaporated in rotary evaporator (Buchi R-
114) and then under high vacuum to give 15 g crude
compound. The crude extract was purified by column
chromatography and thin layer chromatography (TLC)
(Merk Ltd.) using petroleum ether-ethyl acetate (30:1 fi
1:30) as running solvent system.
Optimization of media
Standardization of basal media
Standardization of media for optimum growth and bioac-
tive metabolite production leaf extract, soil extract, NB and
PDB media were used. After 10 days of incubation at 25C
and pH 6.4, the biomass accumulation and secondary
metabolite production was recorded. Biomass accumula-
tion was determined by drying the mycelial mat at 70C
until a constant weight was obtained and expressed as mg/
ml. Based on the optimum growth and bioactive metabolite
production leaf extract was selected as basal medium for
further optimization.
Estimation of bioactive metabolite
The k-max in ethyl acetate of the purified active compound
was determined by using a UV spectrophotometer (SPE-
CORD 210). A standard curve of the bioactive compound
was calibrated and the concentrations of the bioactive
compound produced were determined by comparing the
optical density at 270 nm.
Effect of supplementary Carbon and Nitrogen source
Various carbon sources such as glucose, dextrose, starch,
lactose, mannitol, sucrose, glycerol and nitrogen sources
such as beef extract, yeast extract, peptone, (NH4)2SO4,
NH4Cl, respectively were amended separately into the
basal medium (leaf extract) at a concentration of 0.1%. The
strain DF2 was inoculated to the respective media and
incubated at 25C for 10 days in stationary condition and
their respective biomass vis-à-vis metabolite production
were recorded.
81
Effect of Incubation period
The strain DF2 was inoculated into the basal medium (leaf
extract) amended with dextrose and incubated up to 15
days in stationary condition. Their growth and the sec-
ondary metabolite production was determined everyday by
comparing mycelial weight and UV k-max of the clear
supernatant.
Effect of Incubation Temperature
The strain DF2 was inoculated into basal medium amended
with dextrose and grown in various ranges of temperature
starting from 10 to 50C at a difference of 5C for 10 days.
Effect of Initial pH
The various initial pH ranges were adjusted from 3 to 11 at
a difference of one to the basal media amended with dex-
trose and DF2 was inoculated for 10 days. The biomass and
bioactive metabolite production were estimated by com-
paring the UV k-max of the clear supernatant.
Effect of Amino acid
The effect of various amino acids on growth and bioactive
metabolite production was compared in basal medium in
combination with three carbon sources glucose, dextrose and
sucrose. The amendment of amino acids and carbon sources
concentrations were 0.1 mg/ml and 0.1% respectively. The
strain DF2 was inoculated to the media and incubated for 10
days at 25C in stationary condition. The biomass accu-
mulation and bioactive metabolite production for each
media composition were estimated as mentioned above.
Effect of NaCl concentration
The effect of salinity on the growth and bioactive metabo-
lite production was carried out by incubating the fungus in
various concentration of NaCl ranging from 1% to 10%,
into the basal media amended with 0.1% dextrose. The
biomass accumulation and bioactive metabolite production
for each NaCl concentration were estimated as stated above.
Specific rate of product formation (qp)
The specific rate of active metabolite production (qp) was
calculated according to the following equation:
qp ¼ 1=XðdP=dtÞ;
Where X is the biomass concentration (lg/ml), P is
antimicrobial agent concentration and t is time respectively.
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The derivative dP/dt was calculated according to the
method proposed by Le Duy and Zajic (1973) and software
for graphical analysis Origin PRO-7.5.
Statistics
scrapped out and bioassayed to determine the active
fraction. The pure compound was soluble in ethyl acetate,
methanol and sparingly soluble in water. The active frac-
tion with Rf value 0.52 showed UV k-max in ethyl acetate
at 270 nm. (Fig. 1)

The experiments were conducted in completely random- Determination of MIC

ized block design and subjected to analysis of variance to
test the null hypothesis. Duncans multiple range test
(DMRT) was done to compare that the sample means were
significantly different from each other at a significant level
of P > 0.01 (Gomez and Gomez 1984).
Results and discussion
Characterization of DF2
Colonies of the fungus DF2 are initially white on PDA
medium and slowly turn into dark green to dark brown
while reverse is off-white with wavy edges. Under micro-
scope the hyphae are septate and hyaline. Conidiophores
are medium length, simple or branched. Microconidia are
abundant, single-celled and clavate. Macroconidia are very
rare. The 18s rRNA gene sequence homology of DF2
showed 93% similarities by NCBI blast service with the
most closely related species Fusarium proliferatum
AY762371.1. From these characteristics the genus of DF2
can be identified as Fusarium sp. belongs to the fungal
class Deuteromycetes and order Fusarial.
Characterization of the bioactive metabolite
MIC of bioactive compound was defined as the lowest
concentration at which there was 100% inhibition of
growth compared to antibiotic free control. Lowest MIC of
20 lg/ml was found against F. semitectum and F. oxy-
sporum and highest MIC of 40 lg/ml was found against
P. aeruginosa (Table 2). Active compound revealed high-
est inhibition zone of 27 mm against F. oxysporum and
lowest inhibition zone was against P. aeruginosa (15 mm).
Similar MIC (25 lg/ml to 100 lg/ml), as well as inhibition
zone, produced by some endophytes were reported by
many workers (Gary et al. 2004; Lu et al. 2000).
Optimization of media
Even though potato dextrose media was used for the iso-
lation of Fusarium sp. DF2, further standardization of
media showed that leaf extract was suitable to use as basal
medium (Fig. 2). Comparative study of growth vis-à-vis
secondary metabolite production indicated a statistically
significant higher biomass and bioactive metabolite pro-
duction in leaf extract by Fusarium sp. DF2. Hence, leaf
extract media was used to optimize different cultural and
environmental parameters for growth and bioactive sec-
ondary metabolite production.

Approximately ~ 500 mg/l crude extract was obtained
from the culture broth in solvent extraction. In preparative
TLC six major fractions from the crude compound were
recovered with different Rf values (Table 1). Each of
the major bands from the TLC plates with different Rf was
Effect of carbon and nitrogen
Figure 3 showed the details of the effect of carbon and
nitrogen source on production of biomass and bioactive
metabolite. A statistically significant higher biomass

Table 1 In vitro antibiosis of

Test organisms Rf values (Inhibition zone diameter in mm)
the six fractions recovered from
the TLC of crude bioactive 0.98 0.82 0.71 0.52 0.32 0.11
compound against the test
organisms Bacillus Subtilis – – – 17 (±2) – –
Staphylococcus aureus – – – 19 (±2) – 15
Mycobacterium smegmatis – – – 15 (±2) – –
Mycobacterium phlei – – – 17 (±2) – –
Escherichia coli – – – 20 (±2) – –
Klebsiella pneumoniae – – – 16 (±2) – –
Pseudomonas aeruginosa – – – 15 (±2) – –
Candida Albicans – – – 21 (±2) – 17
Fusarium oxysporum – – – 25 (±2) – –
Concentration of the Fusarium semitectum – – – 24 (±2) – –
fractions = 100 lg /ml

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World J Microbiol Biotechnol (2008) 24:79–87 83

Fig. 1 Standardization UV k-max in ethyl acetate of the pure

bioactive compound isolated from Fusarium sp. DF2
Fig. 3 Effect of carbon and nitrogen on growth and production of
bioactive metabolite by Fusarium sp. DF2
Table 2 Determination of minimal inhibitory concentrations (MIC)
of the secondary metabolite isolated from the strain Fusarium DF2
Test organisms Crude extract Inhibition MIC of pure (2.1 lg/ml), respectively showed significantly less amount
zone diameter in mm compound of biomass and bioactive metabolite production.
(1.00 mg/ml) (in lg /ml)
Amendment of yeast extract (15.7 lg/ml) enhanced the
Bacillus subtilis 19 25 (±2) secondary metabolite production while beef extract
Staphylococcus aureus 20 25 (±2) (2.5 mg/ml), (NH4)2SO4 (2.3 mg/ml) and peptone (1.6 mg/
Mycobacterium smegmatis 16 35(±2) ml) increase biomass production but not bioactive metab-
M. phlei 15 30(±2) olite. Radu et al. (2002) also reported the maximum
Escherichia coli 21 25(±2) production of antifungal and antitumour compound from
Klebsiella pneumoniae 17 35(±2) endophytic fungi grown in a glycerol (4%) and yeast ex-
Pseudomonas aeruginosa 15 40(±2) tract (0.5%) amended media. The simple carbohydrates
Candida albicans 19 30(±2) like glucose, dextrose through metabolic pathway affect on
Fusarium oxysporum 27 20(±2) production of intermediates leading to primary as well as
F. semitectum 25 20(±2)
secondary metabolites in addition to CO2, water and energy
(Turner 1971). Addition of glucose resulted highest growth
Data were mean observation of three independent experiments of the fungus, but significantly in many fermentation pro-
cesses higher concentration of glucose has a suppressive
(3.7 mg/ml) and bioactive metabolite (16 lg/ml) were effect on production of bioactive metabolites (Hutter
produced by the strain DF2 in dextrose amended media and 1982).
it was followed by glucose, sucrose and glycerol, respec-
tively in comparison to control (10.5 lg/ml) basal medium. Effect of temperature, initial pH and incubation period
Starch (3.4 lg/ml), lactose (2.3 lg/ml) and mannitol
Maximum growth (3.2 mg/ml) and bioactive metabolite
production (16.4 lg/ml) by Fusarium sp. DF2 was re-
corded at incubation temperature 25C (±2) and it was
followed by 30C, 35C, respectively (Fig. 4). Less growth
and bioactive metabolite production was found at low
(15C) as well as in high temperature (40C). An expo-
nential growth pattern was recorded at temperature
between 10C to 30C, while growth ceased at < 10C and
> 45C. Study proved that low temperature may cease the
metabolic activity of the fungus and high temperature kills
the cell of the fungus. Similarly, Huang et al. (2001) also
reported the isolation of antifungal and antitumour agent
from endophytic fungi at 25C and 7 days incubation.
Initial pH 6 of the medium was found to be the opti-
Fig. 2 Optimization of basal medium for the strain of Fusarium mal for growth (3.5 mg/ml) and bioactive metabolites

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Fig. 4 Effect of temperature on growth and bioactive metabolite

production by Fusarium sp. DF2
Fig. 6 Effect of incubation period on growth and bioactive metab-
olite by Fusarium sp. DF2
production (14.3 lg/ml) by Fusarium sp. DF2 (Fig. 5).
Neutral pH also supports the growth (3.2 mg/ml) and
day of incubation. Maximum growth and production of
bioactive metabolite production (13.2 lg/ml) of the strain.
antimicrobial agent was recorded after the fungus reached
No growth was observed at pH <3 and pH >11. Digrak
its stationary phase and remaining almost constant upto 15
et al. (2001) reported that highest production of biomass by
day. Stinson et al. (2003) also reported the same result in
F. equiseti was at pH 8, whereas maximum toxic metab-
case of endophyte Gliocladium sp. The highest value
olite was produced at the pH 5. The pH of culture media is
of specific rate of bioactive metabolite formation
one of the determining factor for the metabolism and hence
(0.0058 day–1) was observed on 3rd day (Fig. 7), and it was
for the biosynthesis of secondary metabolites. The pH is
followed by 5th day (0.0041 day–1).
related to permeability characteristics of the cell wall and
Comparison of the effect of NaCl concentration, 2.5–
membrane and thus has got effect on either ion uptake or
3.0% was found to be optimum for growth (4.3 mg/ml,
loss to the nutrient medium (Hansen 1968).
3.8 mg/ml) and production of the antimicrobial agent
Although literature exists on the growth of fungus in
(10.6 lg/ml, 10.1 lg/ml), while concentration above 6%
acidic conditions, in our study it was found that Fusarium
reduces the growth as well as the metabolite production
sp. DF2 grows in between pH 6 to near neutral. Rubini
(Fig. 8).
et al. 2005 also reported the growth and anitimicrobial
agent production at neutral pH.
Effect of different amino acid on growth and production
The incubation of 9 to 10 days was observed to be
of secondary metabolites
optimum for maximum biomass (3.3 mg/ml) and bioactive
metabolite (14.4 lg/ml) production (Fig. 6). The growth
Amino acids added with three carbon sources, dextrose in
and secondary metabolite production remain static after 10
combination with glutamic acid enhances the growth

Fig. 5 Effect of pH on growth and bioactive metabolite production

by Fusarium sp. DF2 Fig. 7 Specific rate of product formation (qp) by Fusarium sp. DF2

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Fig. 8 Effect of salt concentration on growth and bioactive

metabolite production by Fusarium sp. DF2
Fig. 10 Effect of different amino acids in combination with glucose
on growth and bioactive metabolite production by Fusarium sp. DF2
(3.2 mg/ml) whereas phenyl alanine in combination with
dextrose enhances the bioactive metabolite production
(15.4 lg/ml). On the other hand although aspartic acid,
alanine, histidine, methionine, threonine promoted the
growth but the bioactive metabolite production was insig-
nificant (Fig. 9).Similarly the amendment of methionine in
combination with glucose increased both biomass (3.5 mg/
ml) and antimicrobial agent (13.2 lg/ml) production in
comparison to the control (Fig. 10). Except methionine
none of other amino acids used were found to be effective
in enhancing the production of bioactive metabolite in
combination with glucose.
The maximum growth (3.3 mg/ml) as well as bioactive
metabolite production (12.5 lg/ml) was recorded in the
combination of methionine and sucrose (Fig. 11), followed
by the control (10.5 lg/ml), Glycine (8.2 lg/ml) and
Glutemic acid (7 lg/ml), respectively. It reflects that amino
acid supplement may have some role by sharing their Fig. 11 Effect of different amino acids in combination with sucrose
on growth and bioactive metabolite production by Fusarium sp. DF2
carbon ring or both carbon and nitrogen skeleton in to the

primary or secondary metabolism processes of microor-

Fig. 9 Effect of different amino acids in combination with dextrose
on growth and bioactive metabolite production by Fusarium sp. DF2
ganisms (Noaman et al. 2004). Kim et al. (2005) reported
the importance of rice oil for improve production of
cephalosporin-C production by Cephalosporium acremo-
nium M25.
The endophytic fungus Fusarium sp. DF2 isolated from
T. wallichiana, showed potential activity against the tested
microorganisms in vitro. The inhibition zones found in vi-
tro >15 mm and MIC against virulent test pathogen have
well-evinced to forecast the potential of the strain to use for
antimicrobial agent production. Many studies on isolation
and characterization of endophytic diversity from different
medicinally important plant species have been done or are
in progress round the world (Johri 2006). Some workers
have given a brief record on antifungal and antibacterial
activity of endophytes isolated from different gymno-
sperms of North East India (Bala et al. 1999; Saikia et al.

123
86
2005). Though many studies were being taken up but the
strain isolated from T. wallichiana of Arunachal Pradesh is
yet to report in detail.
Conclusion
From the above observations it can be concluded that due
to the wide antimicrobial activity of the strain Fusarium sp.
DF2, further exploration of the strain both at biochemical
and molecular level along with large scale field application
can be exploited for pharmaceutical as well as biotechno-
logical application.
Acknowledgements We are grateful to Dr. P.G. Rao, Director,
RRL, Jorhat, Assam, India for providing the facilities and CSIR,
Govt. of India for providing the fellowship to conduct the work
References
Adinarayana K, Prabhakar T, Srinivasulu V, Rao AM, Jhansi LP,
Ellaiah P (2001) Optimization of process parameters for
cephalosporin C production under solid state fermentation from
Acremonium chrysogenum. Process Biochem 39:171–177
Ainsworth GC, Sparrow FK, Sussman AS (1973) The fungi, an
advanced treatise. A taxonomic review with keys: ascomycetes
and fungi imperfecti, Vol IVA. Academic Press, New York,
pp 105–514
Bala S, Uniyal GC, Chattopadhyay SK, Tripathi V, Sashidhara KV,
Kulshrestha M, Sharma RP, Jain SP, Kukreja AK, Kumar S
(1999) Analysis of taxol and major taxoids in Himalayan yew,
Taxus wallichiana. J Chromat 858:239–244
Basak K, Majumdar SK (1975) Mineral nutrition of Streptomyces
kanamyceticus for kanamycin formation. Antimicrob Agent
Chemot 8(4):391–395
Digrak M, Eluk SZ (2001) Determination of some fungal metabolite
as influenced by temperature, time, pH and sugars by bioassay
method. Turk J Biol 25:197–203
Glienke-Blanco C, Aguilar-Vildoso CI, Carneiro Vieira ML, Vianna
Barroso PA, Azevedo JL (2002) Genetic variability in the
endophytic fungus Guignardia citricarpaisolated from citrus
plants. Gene Mol Biol 25(2):251–255
Gomez A, Gomez A (1984) A statistical procedure for agricultural
research. 2nd edn. John Willy and Sons, Kwanchi, pp 188–191
Griffiths DJ, Saker ML (2003) The Palm island mystery disease 20
years on: a review of research on cyenotoxin cylindrospermop-
sin. Inc Environ Toxicol 18:78–93
Hawksworth DL, Sutton BC, Ainsworth GC (1983) Dictionary of the
fungi, 7th edn. Common Wealth Mycological Institute, Kew,
Surrey, pp 1–500
Hansen OH (1968) Ecology, physiology, and biochemistry of blue-
green algae. Annu Rev Microbiol 22:47–57
Huang Y, Wang J, Li G, Zheng Z, Su W (2001) Antitumor and
antifungal activities in endophytic fungi isolated from pharma-
ceutical plants Taxus mairei, Cephalataxus fortunei and Torreya
grandis. FEMS Immunol Med Microbiol 31:163–167
Hutter R. (1982) Design of culture media capable of provoking wide
gene expression. In: Bullock Jd, Nisbet LJ, Winstanley DJ (eds)
Bioactive microbial products, Search and Discovery. Academic
Press, London, pp 37–50
123
World J Microbiol Biotechnol (2008) 24:79–87
Johri BN (2006) Endophytes to the rescue of plants. Curr Sci
90(10):1315–1316
Kang JG, Shin SY, Kim MJ, Bajpai V, Maheshwari DK, Kang SC
(2004) Isolation and anti-fungal activities of 2-Hydroxymethyl-
chroman-4-one produced by Burkholderia sp. MSSP J Antibio
57(11):726–731
Kim RN, Lim SJ, Hong SI, Kim SW (2005) Optimization of feed
conditions in a 2.5-I fed-batch culture using rice oil to improve
cephalosporin C production by Cephalosporium acremonium
M25. World J Microbiol Biotechnol 21:787–789
Le Duy A, Zajic JE (1973) A geometrical approach for differentiation
of an experimental function at a point: applied to growth and
production. Biotechnol Bioeng 15:805–815
Lethimaki J, Moisander P, Sivonen K, Kononen K (1997)
Growth, nitrogen fixation and nodularin production by two
Baltic Sea cyenobacteria. Appl Environ Microbiol 63(5):1647–
1654
Lu H, Zou WX, Meng JC, Hu J, Tan RX (2000) New bioactive
metabolites produced by Colletotrichum sp., an endophytic
fungus in Artemisia annua. Plant Sci 151:67–73
Myers N, Russel AM, Cristina G, Gustavo-Foneca AB, Kent J (2000)
Biodiversity hotspots for conservation priorities. Nature
403:853–858
Mc Afee BJ, Taylor A (1999) A review of the volatile metabolites of
fungi found on wood substrates. Nat Toxins 7:283–303
Moita C, Feio SS, Nunes L, Joa M, Curto M, Roseiro JC (2005)
Optimisation of physical factors on the production of active
metabolites by Bacillus subtilis 355 against wood surface
contaminant fungi. Int Biodeter Biodegrad 55:261–269
Noaman NH, Fattah A, Khaleafa M, Zaky SH (2004) Factors
affecting antimicrobial activity of Synechococcus leopoliensis.
Microbiol Res 159:395–402
Pereira JO, Azevedo JL, Petrini O (1993) Endophytic fungi of
Stylosanthes: a preliminary study. Mycologia 85:362–364
Pereira JO, Vieira MLC, Azevedo JL (1999) Endophytic fungi from
Musa acuminata and their reintroduction in axenic plants. World
J Microbiol Biotechnol 15:43–46
Radu S, Kqueen CY (2002). Preliminary screening of endophytic
fungi from medicinal plants in Malayasia for antimicrobial and
antitumour activity. Malaysian J Med Sci 9(2):23–33
Regina M, Santos G, Edson RF (2002) Meroterpenes from Penicil-
lium sp. found in association with Melia azedarach. Photochem
61:907–912
Rubini MR, Rute TSR, Pomella AW, Cristina SM, Arajo LW, Santos
DRD, Azevedo JL, (2005). Diversity of endophytic fungal
community of cocao (Thebroma cacao L.) and biological control
of Crinipellis perniciosa, causal agent of Witches’ Broom
disease. Int J Biol Sci 1:24–33
Saikia D, Khanuja SPS, Shasany AK, Darokar MP, Kukreja AK,
Kumar S, (2005). Assessment of diversity among Taxus
wallichiana accessions from northeast India using RAPD
analysis. PGR Newslett FAO-IPGRI 121:27–31
Sailer M, Helms GL, Henkel T, Niemcruzura WP, Stiles ME, Vederas
JC (1993) 15-N- and 13 C- labelled media from Anabaena sp. for
universal Isotopic labeling of NMR resonance assignment of
leucin A from Leuconostoc gelidum and nisin A from Lacto-
coccus lactis. Biochem 32(1):310–318
Schillinger U, Lucke FK (1989) Antibacterial activity of lactobacillus
sake isolated from meat. Appl Environ Microbiol 55(8):1901–
1906
Selvin J, Joseph S, Asha KRT, Manjusha WA, Sangeetha VS,
Jayaseema DM, Antony MC, Vinitha AJD (2004) Antibacterial
potential of antagonistic Streptomyces sp. isolated from marine
sponge Dendrilla nigra. FEMS Microbiol Lett 50:117–122
Stinson M, Ezra D, Hess WM, Sears J, Strobel G (2003) An
endophytic Gliocladium sp. of Eucryphia cordifolia producing
World J Microbiol Biotechnol (2008) 24:79–87
selective volatile antimicrobial compounds. Plant Sci 165:
913–922
Stierle A, Strobel G, Stierle D (1993) Taxol and taxane production by
Taxomyces andreanae, an endophytic fungus of Pacific yew.
Science 260:5105, 214–216
Strobel G, Bryn D (2003) Bioprospecting for microbial endophyte
and their natural products. Microbiol Mol Biol Rev 67(4):491–
502
Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from
endophytic microorganisms. J Nat Prod 67(2):257–268
Strobel G, Yang X, Sears J, Kramer R, Sidhu RS, Hess WM (1996)
Taxol from Pestalotiopsis microspora, an endophytic fungus of
Taxus wallachiana. Microbiology 142:435–440
87
Turner WB (1971) Fungal metabolite. Academic Press, London and
New York
Vahidi H, Kobarfard F, Namjoyan F (2004) Effect of cultivation
conditions on growth and antifungal activity of Mycena lepto-
cephala. Affr J Biotechnol 3(11):606–609
Wang J, Huang Y, Fang M, Zhang Y, Zheng Z, Zhao Y, Su W (2002)
Brefeldin A, a cytotoxin produced by Paecilomyces sp. and
Aspergillus clavatus isolated from Taxus mairei and Torreya
grandis. FEMS Immunol Med Microbiol 34:51–57
Wang J, Li G, Lu H, Zheng Z, Huang Y, Su W (2000) Taxol from
Tubercularia sp. strain TF5, an endophytic fungus of Taxus
mairei. FEMS Microbiol Lett 193:249–253
123