MATERIALS AND METHODS

Experimental studies were undertaken during monsoon, winter and summer seasons
during 2011 to 2014 in the Karnatak University Botanical Garden Dharwad -580003, India.
Dharwad district is situated in the Western sector of the northern half of Karnataka State
shown in (Fig. 2). Geographically the selected area is situated in between 15°02' and 15°51'
North longitudes and 73°43' and 75°35' East latitude. The temperature is usually 20.2°C in
June and high as 34.42°C in March. The annual rain fall is 600-850 mm. The climate is
usually semi humid to humid. Soil is covered with a hard, compact crust having dark brown
colour.

The Karnatak University Botanical Garden was laid out in the year 1963 in an area of
about 1, 61,874 Sq Meters. The site commands an elevation of 2,700 ft. above sea level. The
garden has been established under different subdivisions, as the systematic, the Economic
plants, the Herb Garden, the Shade Garden, the Fruit Plot, the Vegetable plot, the lotus pond
etc. Nearly 150 families have been so far represented. It is expected that the gardens would
prove useful to the Botany Students of the University including the affiliated colleges and
also the interested public of the area. It is interesting to note that this is the only botanical
garden in the northern western part of the state of Karnataka.

Botanical gardens are important centers for education and collaboration of their
activities with the University and Research Institutes. Karnatak University Botanical Garden
were conceived and established for teaching of Botany, Medicinal and Horticultural plants
which dates back to 16th century. Botanical gardens comprise arboretum, herbal garden,
conservatories, green houses, demonstration of experimental plots, aquatic bodies, etc.
Incredibly diverse collections from temperate, sub-temperate, tropical sub-tropical and desert
regions, well documented and aesthetically displayed plants species can offer an extensive
range of opportunities to children, college students, researches and general public. They lead
themselves to teach about plant diversity and their habitats, functions of plant and plant
classifications, floral designs, conservation, economic uses of the plants, horticultural
operations and bioaesthetic planning.
Fig 2: Showing the list of experimental plants growing in Karnatak University Botanical

Garden Dharwad.

1. Garcinia indica L.

2. Saraca indica L.

3. Piper betel L.

4. Basella rubra L.

5. Vitis quadrangularies L.

6. Mimosa pudica L.

7. Oxalis corniculata L.

8. Ruta graveolens L.
PLATE-1. Showing the Photographs of four selected medicinal plants for the study of
fungal endophytes

A. Garcinia indica L.
B. Saraca indica L.
C. Piper betel L.
D. Basella rubra L.
PLATE-2. Showing the photographs of four selected medicinal plants for the study of
fungal endophytes

E. Vitis quadrangularies L.
F. Mimosa pudica L.
G. Oxalis corniculata L.
H. Ruta graveolens L.
Collection of plant samples
A total of eight medicinal plants have been selected for the present investigation.
Among these two are trees namely, Garcinia indica L., Saraca indica L., two are climbers,
these are Piper betle L., Basella rubra L., two are shrubs, these are Vitis quadragularis L.,
Mimosa pudica L., and two herbs are Oxalis corniculata L., Ruta graveolens L. (Theodore
cooke 2006., Olasantan et al., 2008., Srivastava et al., 2012).
Healthy and matured leaves of these eight medicinal plants were selected and plant
leaves were collected randomly from each medicinal plant. For each plant sample was kept in
polythene bags with labeled, sealed and brought to the laboratory within 12 hrs and stored at
40C in deep freezer until isolation procedures were completed according to the procedure of
Kumar and Hyde, 2004.

Isolation of endophytic fungi:

The endophytic fungi were isolated following the method of (Dobranic et al., 1995,
Kumaresan and Suryanarayanan, 2002). The collected samples were washed under running
tap water so as to remove epiphyllous debris followed by washed thoroughly in double
distilled water and cut into (0.5-1cm) segments using sterilized scissor. Five selected pieces
of each experimental plant leaves were sterilized by dipping in 75% ethanol for 1 min,
Sodium hypochlorite solution (3.25%) for 3 min (2% available chlorine), ethanol (75%) for
30 sec and washed thoroughly 3 to 4 rinse of double distilled water and blotted with sterile
tissue paper. Incubated leaf segments are shown in Plate-3.

Efficacy of surface sterilization:

The effectiveness of sterilization was checked by placing the sterilized leaf segments
on the surface of PDA medium, following the procedure of (Schulz et al., 1993). When, the
growth of fungi was not seen on the medium after 7 days, there will be an indication that
sterilization procedure was effective in removing the surface fungi. Later on sterilized leaf
segments were pressed, on to the surface of the PDA medium. The absence of growth of any
fungi on the medium after 1- 12 days confirmed that endophytic isolations were only from
the internal tissues of the samples used. Similarly, the surface sterilization procedure was
adopted to remove the surface fungi according to the procedure of (Schulz et al., 1993).
PLATE-3. Showing the photographs in eight medicinal plants leaf segments inoculated
on PDA media.

A. Photograph showing the growth of endophytic fungi from G. indica L.

B. Photograph showing the growth of endophytic fungi from S. indica L.
C. Photograph showing the growth of endophytic fungi from P. betle L.
D. Photograph showing the growth of endophytic fungi from B. rubra L.
E. Photograph showing the growth of endophytic fungi from V. quadragularies L.
F. Photograph showing the growth of endophytic fungi from M. pudica L.
G. Photograph showing the growth of endophytic fungi from O. corniculata L
H. Photograph showing the growth of endophytic fungi from R. graveolens L.
PLATE - 4. A to F showing the photograph of pure cultures of fungal endophytes from the

leaf samples of experimental plants.

G-H. Showing the photograph of pure culture test tube slants stored on PDA

media.
PLATE – 4A., A-E. Showing the photograph of pure cultures of fungal endophytes from the

leaf samples of eight experimental plants.

F. Showing the photograph of pure culture test tube slants stored on PDA media
Incubation of experimental specimens
Surface sterilized leaf segments were transferred to sterilized Petri-plates (90mm
diam) containing Potato Dextrose Agar (PDA), according to (Dreyfuss, 1986; Bills and
Polishook, 1992), the PDA composed with 1% Streptomycin Sulphate (100mg/L) this
antibiotic inhibit bacterial contamination. All the plates were labeled and incubated at
25±2°C incubated leaf segments were observed every day for the growth of endophytic fungi
until fungal growth appeared (Lacap et al., 2003). Fungal growth from the plant tissues were
screened with the help of stereo - binocular microscope and again transferred on to fresh
PDA medium. After purifying these isolates for several times, final pure cultures were
transferred to PDA slants in test tubes (Shown in Plate-4 and 4A) and maintained at 40c in
refrigerators.

Identification of fungal endophytes

For characterisation of the morphology of fungal isolates, slides were prepared from
cultures and they were stained with lactophenol blue reagent and examined under a bright-
field and phase-contrast microscope, identified by referring standard manuals (Barnett and
Hunter, 1972; Subramanian, 1983; Sutton, 1980; Joseph C. Gilman 2001; Nagamani et al.,
2006), monographs and research articles viz; Ainswarth et al., (1973), Bilgrami et al., (1979,
1981, 2004), Blodgett et al., (2000), Devarajan and suryanaranan (2006), Krohn et al., (2007)
Li and Strobel (2001), Photitia et al., (2005) Suryanarayanan et al., (1998, 2002), Tejasvi et
al., (2007). Identification was done based on morphological characteristics such as growth
pattern, hyphal characters, colour of colonies on the medium and type of media, surface
texture, margin character, aerial mycelium, mechanism of spore production and
characteristics of the spores. Measurements of all the fungal characters were made in water
mounts, and the slides were prepared and mounted in lactophenol and sealed with DPX
mountant. All experiments and observations were repeated at least thrice. Finally
photography and measurement of fungi was taken from fluorescent microscope shown in
plate (Plate 10F). Author and publication citations for following isolated endophytic fungi
from Karnatak University Botanical Garden Dharwad are abbreviated according to http:/
www. Indexfungorum. Org/ Authors of fungal names. Htm and Index Fungorum (www.
Species fungorum; accessed 2014). All the isolated and identified fungi were stored in
microbiology laboratory Karnatak University Dharwad (MLKUD).
Methods used for sporulate sterile fungi:
 Culture samples that failed to sporulate which were again repeated on different
nutrients media. Those endophytes which were not sporulated on PDA (200g potatoes, 20g of
dextrose, 20g of Agar powder) MEA (MEA = 30g malt; 2g agar; 1000ml distilled water) and
OMA (OAT = 75g Oatmeal; 2g Agar; 1000ml distilled water) medium respectively, were
considered as sterile fungi. Such fungi were categorized separately and given a code based on
colour of mycelium and morphological feature of colony.
Seasonal variation of fungal endophytes

The isolated endophytic fungi were screened for colonization rate, to understand the
total colonization frequency and distribution of species in different seasons by using
appropriate statistical packages.
Percentage of colonization rate was calculated according to the formula proposed by
Devarajan et al., (2002).

Percentage of colonization rate = Number of segments colonized by any fungi X 100

Total number of segments incubated

Frequency of occurrence of dominant endophytic fungi was calculated (Devarajan et al.,

2002).

Percentage of colonization frequency = Total no of segments yielding given fungus X 100

Total number of segments incubated

Examination of fungal endophytes

The most dominant endophytic fungi in all the eight selected medicinal plants were
isolated and the most predominant endophytic fungi are; Alternaria chlamydospora Mouch.,
Aspergillus awamori Var. hominis Bat & maia., Aspergillus flavipes (Bainier & Sartory)
Thom and Church., Bipolaris nodulosa (Sacc.) Shoemaker., Nigrospora sphaerica (Sacc.) E.
W. Mason., Nigrospora padwickii Prasad, Agnihotri & J. P. Agarwal., Penicillium citrinum
Thom., Rhizopus nigricans Var. Minutus chaudhuri & Sachar.

Antagonistic studies of fungal endophytes

To study the antagonistic property of endophytic fungi, the most dominant endophytic
fungi in all the eight selected medicinal plants were selected and these are tested against
Colletotrichum capsici (Syd. & P. Syd.) E.J. Butler & Bisby. a pathogen by dual culture
method.
Biocontrol studies on Colletotrichum capsici by using endophytic fungi
Anthracnose disease caused by Colletotrichum species causing reducing marketable
yield from 10% to 80% of the crop production in some developing countries. Anthracnose is
mainly a problem on mature fruits, causing severe losses due to both pre- and post-harvest
fruit decay.
Colletotrichum is one of important plant pathogens, and it occurs in worldwide and
causing anthracnose disease on wide range of hosts including cereals, legumes, vegetables,
perennial crops and tree fruits. Among these hosts, chili (Capsicum spp.), is an important
economic crop of worldwide that is being affected by this endophytic fungi.
Isolation of Colletotrichum capsici

Infected leaves and fruits were carried out from the collection of chilies growing
district of Haveri Karnataka. Infected leaves and fruits were cut into 2-3cm size and placed in
the moisture chamber apparatus and incubated at room temperature 260c for 3-5 days. After
the incubation period, mycelia growth from the infected portion was subculture onto PDA
media and after confirming firmly as Colletotrichum capsici, then it’s pure culture was
maintained on PDA slants in the refrigerator at 4°C. For cross examination of pathogen,
strain also collected from Microbiology Department of University Agricultural Science
Dharwad and compared with the isolated pathogen.
Screening for antifungal activity
Laboratory experiments were carried to examine the antagonistic efficacy of
endophytic fungi. Five days old mycelia discs (5mm diam) of test pathogens were placed at
the centre of the petri plate containing PDA medium. Three 5mm discs of the endophytic
fungi were inoculated at the 3 sides of the centrally placed pathogen disc (Naik et al., 2006).
In control studies, one set was inoculated with selected pathogens for comparison of radial
growth with dual culture plates.
The plates were incubated for 7-12 days at room temperature and radial growth of the
pathogen was measured with comparing to the control sets (measurement was taken with help
of inhibition zone scale). Percentage inhibition of the pathogen by the endophyte was
calculated using the formula I=(C-T)/C X 100 following the procedure of (Joseph et. al.,
2003), where C is the radial growth of the pathogen (mm) in the control and T is growth of
pathogen (mm) in the test. Results were recorded for inhibition zone in triplicates.
Mass cultivation of the fungus for GC- MS (Gas Chromatography Mass-Spectrometry)
analysis
Isolated, identified endophytic fungi and with significant antifungal activity shown
Nigrospora sphaerica were processed for further GC-MS analysis, in Erlenmeyer flasks
containing Potato Dextrose Broth. The fungus inoculated flasks were incubated at room
temperature (26±20C) under static conditions for 21 days.
Extraction and isolation of the bioactive compounds
After incubation, fungal mycelia of Nigrospora sphaerica were separated from liquid
culture media and soaked in HPLC grade Methanol in an overnight. The cells were disturbed
using mortar and pestle for 10 min followed by filtration in Whatman Number 1 filter paper.
The methanol extract of endophyte was separated after 3-5 times filtration process.
GC-MS analyses
The bioactive crude extract was separated into various fractions by column
chromatography. GC-MS analyses were performed at University Science Instrument Centre
(USIC), Karnatak University Dharwad, India. GC-MS measurements were performed with a
Shimadzu instrument equipped with GC: Aligent QP 2010,GC- MS detector 5975C,
Ionization for MS: Electron Impact Ionization, Mass Analyzer: Quadrupole, Software: Real
time, Library: NIST 2008, column: HP 5 ms, Dimensions: 30m L X 0.25mm ID x 0.25μm
film thickness, initial temperature is 0 to 400C 2 min hold time, ram temperature is 1000C to
2800C 30 min, carrier gas is helium, flow (ml/min) is 1.0, split flow: 1ml/min 1:1, injection
volume: 1μl, Scan mass range: 40 m/z-500m/z and polarity +ve. GC-MS analysis was
performed based on the database having more than many patterns. The spectrum of the
unknown compound was compared with the spectrum of the known compounds from the
library based on retention time and mass spectra (NIST - library). (Govindappa et al., 2014).
In GC-MS, 15 peaks were observed in the fraction of 6 of methanol extract of
Nigrospora spp. based on retention time and chromatograms using the library, identified
compounds are listed in (Table-36). The compounds have already reported as strong
antioxidant molecules and were isolated from different natural sources.

Interaction studies between Arbuscular Mycorrhizal fungi and Nigrospora sphaerica

Collection of soil sample
Soil sample from the root zone of Ocimum sanctum Linn. was collected. It was done
by digging out by soil digger, a small amount of soil close to the plant roots up to the depth of
5-30 cm.
Isolation of AM spores:
Isolation of AM fungal spore was done by using wet-sieving and decanting technique
following the procedure of (Gerdemann and Nicolsan, 1963) and quantification of AM spores
was done by gridline intersect method (Adholey and Gaur, 1994).
Colonization of AM fungi:
AM fungal root colonization was studied by rapid clearing and staining method
(Phillips and Hayman, 1970). Per cent of AM fungal root colonization was calculated by
using the formula
Percent AM root colonization = No. of root segments infected X100
Total no. of root segments studied

Identification of AM spores:
For identification of AM spores the following criteria was used like conventional
morphological characters i.e. Color, size, shape, wall structure, surface structure/
ornamentation of spores, size of subtending hyphae, hyphal attachement and bulbose
suspensor. The AM spores were identified by using keys of (walker 1983; Schenk and Perez
1990; and Mukherji 1996).
Mass multiplication of AM spores:
Dominant AM fungal spores i.e. Glomus mosseae was isolated from the rhizosphere
of O. sanctum and were mass cultured by using Maize (Zea mays L.) as host plant. After 90
days, soil containing AM spores (50g soil contain approximately 215-245 Chlamydospores)
and colonized roots pieces (10g) were used.
Inoculation :
50g rhizospere soil of host plant (maize) +10g dried AM fungi of G. mosseae
inoculums and the endophytic fungal culture of Nigrospora sphaerica was inoculated at
25±20c for 21 days. Fungal mycelium was harvested and homogenized to break and loosen
the fungal mat. Then it was inoculated (10mg/mL) in soil near the roots of seedlings. The
control was untreated. The pots were kept under normal growth conditions at 25±20c and 12
hrs daylight and were monitored regularly. All the experiments were performed in the
triplicate.
Pot experiments:
Preliminary growth response studies were carried out by using earthnpots measuring
20X30cm (length X breadth) filled with 6Kg contained pure sand: garden soil (1:3) ratio and
its physico-chemical analysis shown (Table 1). Before sowing the O. sanctum seeds all the
pots soil was autoclaved and sterilized three times for 3-consecutive days at the interval of 24
hrs. Similarly Ociumum sanctum seeds were surface sterilized with 2% sodium hypochlorite
solution for 10 min and 70% ethanol for 1 min. finally, the seeds were washed 3-4 times with
double distilled water and sowed in the experimental earthen pots.
Root and shoot biomass:
Shoots and roots were harvested after 90 days after inoculation (DAI). Roots were
washed under tap water to remove adhering soil particles and then both roots and shoots were
cut into small pieces and weighed separately for their fresh weight. These were then oven
dried to a constant weight at 700c and again weighed for dry weight.
Table 1. Physico-chemical characteristics of experimental soil samples used for pot
experiments to study interaction between G. mosseae and Nigrospora sphaerica.

S.N Parameters Results

1. Soil Sandy loam

2. pH (1;2.5) 6.8

3. Conductivity (EC) us/cm 319

4. Moisture (%) 4.76

5. Total organic carbon (%) 1.81

6. Nitrogen (%) 0.08

7. Potassium (%) 7.92

8. Magnesium (%) 0.121

9. Phosphorus (%) 4.50

10. Calcium(%) 0.471

11. Zinc(ppm) 3.85

12. Copper (ppm) 0.034

13. Manganese (ppm) 0.964

14. Iron (ppm) 8.26

Statistical analysis
Data obtained in all the experiments were treated with appropriate statistical tests.
Significance of differences in the frequency of colonization among the host plants and
endophytes was determined by Scheffe’s Post hoc test. Differences in the colonization of
fungi between winter, monsoon and summer seasons were tested by ANOVA.
The data was also subjected for PAST which generates Pearson Correlation Matrix
and Bray-Curtis similarity index to study the correlation between the organisms and
similarity between them respectively.
To describe the taxonomic affinity of endophytic mycota among the various hosts, a
Bray Curtis Similarity Index was used to measure the similarity between pairs of samples
(Arnold et al., 2000). Bray-Curtis similarity was used to describe the taxonomic affinity of
endophytic mycobiota among the hosts (Atmar and Patterson, 1993, 1995).