Full Length Research Paper
Isolation and Identification of Endophytic Fungi from Semecarpus anacardium
Rahul K. Dhabale1[*],
Bhagwan M. Waghmare2 and Suvarna T, Kadam2
1Department of Botany, Bhagwantrao Arts &
Science College, Etapalli Dist. Gadchiroli.India.
2Botany Research Centre, Department of Botany, Maharashtra Mahavidyalaya, Nilanga. Dist. Latur ( MS)
India.
ARTICLE DETAILS ABSTRACT
1. Introduction
Endophytes are live
within plants for at least a part of their life cycle without causing any
visible manifestation of disease (Bacon and white, 2000). A wide range of
plants have now been examined for endophytes, and endophytes have been found in nearly all of them, including
trees, grass, algae and herbaceous plants. Evidence of plant-associated
microorganisms found in the fossilized tissues of stems and leaves has revealed
that endophyte-plant associations may have evolved
from the time higher plants first appeared on the earth (Redecker
et al., 2000). Hawksworth and Rossman
estimated that nearly one million species of endophytes
may exist in the unexplored plants (Strobel and
Daisy, 2003; Arnold, 2005). Endophytes microorganisms
were discovered including fungi, bacteria, and actinomycetes. And fungal endophytes are the most frequently encountered endophytes (Staniek et al.,
2008). It have great promise with diverse potential
for exploitation (Li et al., 2012; Staniek et al., 2008).
An enormous number of different fungi can be isolated from plants growing in
their native habitat. The aim of this research focused on isolate endophytic fungi from roots, stem and leaves of Semecarpus anacardium and
identified by morphological characters.
2.
Materials and
methods
2.1 Plant Sample Collection
Semecarpus
anacardium was collected from Mahur forest Dist. Nanded. All
the samples like root, stem and leaves were collected
from of Semecarpus anacardium.
2.2 Isolation of Endophytic Fungi
Samples
of Semecarpus anacardium like
root, stem and leaves were washed thoroughly in
running tap water. Surface of the plant tissues was treated by following the
methodology of (Murali et al.2007). Plant tissues were immersed in 75% ethanol for 1
minute and in an aqueous solution of sodium hypochlorite (2.5% available
chlorine) for 3 minute, followed by washing
with 70% ethanol for 5 seconds. The tissues were then rinsed in sterile
distilled water and allowed to surface dry under sterile conditions. The
surface-sterilized samples were placed on Petri dishes containing Potato Dextrose
Agar (PDA) (supplemented with streptomycin (100 μg
ml−1) to inhibit bacterial growth) and incubated at temperature at around
28°C.
2.3 Identification of Endophytic Fungi
The isolates of endophytic fungi were identified by the morphology of the
fungal culture, including colony and medium color, Colony characters, Spore
characters, Mycelium characters, Fruiting structures by following the standard
mycological manuals (Barnett and Hunter, 1987; Domsch
and Games, 1993; Sutton, 1980; Nag Raj, 1993). The sterile isolates were grown
on PDA with decoction of host leaves medium to observe sporulation. For
tentative identification, microscopic slides of each endophytic
fungus were prepared and examined under binocular compound microscope for
morphological identification.
3.
Results and
Discussion
From roots, stem and leaves
of Semecarpus anacardium,
in this study yielded 82 isolates (Table. 1). More isolates were obtained from stem
of Semecarpus anacardium
than roots and leaves.
Table.
1 Number of isolates from root stems and leaves of Semecarpus anacardium
|
Host Plant |
Root |
Stem |
Leaves |
Total |
|
Semecarpus anacardium |
18 |
33 |
31 |
82 |
1. Curvalaria lunata
Colonies effuse
hairy and black color on PDA medium. Mycelium immersed in natural substrates.
Conidia with hilum scarcely, remaining smooth-walled, dark brown color. Conidia
predominantly 3-septate, the middle septum below the centre
and the third cell strongly curved, tapering gradually towards the base.
2. Trichoderma
species
Colony color ranged from white, yellowish-green and dark green upon
sporulation. Conidia are mostly green, sometimes hyaline, with smooth or rough
walls and are formed in slimy conidial heads (gloiospora)
clustered at the tips of the phialides.
3. Fusarium species
The colonies
fast growing with discrete sporodochia and white-ochraceous colour. Aerial mycelium
floccose. Macro-conidia abundant and more-celled,
slightly curved or bent at the pointed ends; central part straight,
cylindrical, typically canoe-shaped. Phialides bearing micro-conidia very long.
4. Alternaria alternata
Colonies are fast growing, black to olivaceous-black
or greyish, and are suede-like to floccose. Conidia are obclavate, obpyriform, sometimes
ovoid or ellipsoidal, often with a short conical or cylindrical beak, pale
brown, smooth-walled or verrucose.
5. Aspergillus
species
Colonies
growing rather slow on PDA with creamy-yellow color. Mycelium partly immersed, partly superficial. Stroma none;
Setae and hyphopodia absent. Vesicles small, variable in shape. Conidial heads globose and bright yellow. Conidia globose to subglobose,
smooth-walled, uninucleate, the chains sometimes sliming down.
6. Phomopsis
species
Colonies
grow fast on PDA with abundant floccose, whitish to olivaceous-grey
aerial mycelium; reverse uncoloured. Pycnidia abundantly produced in the centre
of the colonies, olivaceous-brown, dark around the ostiole. Pseudo sclerotia
absent; conidia oblong, two-celled.
7. Eupenicillium ochrasalmoneum
Colonies
show purple-red shade and get dark reddish brown. Clestothecia
are spherical, orange brown in colour, 500µm in diameter. Clestothecia
develops ascospores that are oval or oblong or
spherical, 12-15µm in diameter. Conidiophores originate from branches of hyphae
are hyaline to greenish and septate. Conidia are in
chains and smooth walled.
Table 2.
Numbers of isolates from samples of endophytic fungi
|
Endophytic fungi |
Samples |
Total |
||
|
Root |
Stem |
Leaves |
||
|
Alternaria alternata |
2 |
5 |
3 |
10 |
|
Aspergillus flavus |
3 |
1 |
4 |
08 |
|
Aspergillus niger |
1 |
5 |
5 |
11 |
|
Aspergillus terrus |
- |
4 |
3 |
07 |
|
Aspergillus versicolor |
- |
3 |
3 |
06 |
|
Chaetomium globosum |
- |
1 |
1 |
02 |
|
Curvularia lunata |
4 |
3 |
1 |
08 |
|
Eupenicillium ochrasalmonium |
- |
- |
1 |
01 |
|
Fusarium
equiseti |
2 |
1 |
2 |
05 |
|
Fusarium
oxysporum |
1 |
3 |
3 |
07 |
|
Phomopsis |
2 |
3 |
- |
05 |
|
Trichoderma harzianum |
1 |
2 |
3 |
06 |
|
Trichoderma viridae |
2 |
2 |
2 |
06 |
In this study, Aspergillus niger
had the highest relative frequency (Table.2). Alternaria alternata and Aspergillus flavus
which are frequently identified as endophytes (Masroor Qadri et al., 2013) was
the second most frequent endophytic group. Followed
by Curvularia lunata, Aspergillus terrus, Aspergillus versicolor, Chaetomium globosum, Eupenicillium ochrasalmonium, Fusarium equiseti, Fusarium oxysporum, Phomopsis, Trichoderma harzianum and Trichoderma viridae . These were the dominant genera or order of endophytic fungi found in this study, similar to the
findings reported previously for many tropical endophytic
fungi (Luiz H. Rosa et al., 2012; Nur
Amin et al., 2014; Jane Frohlich, 1999).
4. Conclusion
Eighty two species of endophytic fungi were isolated and identified from roots,
stem and leaves of Semecarpus anacardium
which belong to Alternaria alternata, Aspergillus flavus, Aspergillus niger, Aspergillus terrus, Aspergillus versicolor, Chaetomium globosum, Curvularia lunata, , Fusarium equiseti, Fusarium oxysporum, Phomopsis, Trichoderma harzianum, Trichoderma viridae, 01 unidentified isolates was Eupenicillium ochrasalmonium,
which was identified by molecular method. Aspergillus niger had the
highest relative frequency in this study and Alternaria alternata was the second most frequent endophytic group, followed by Aspergillus terrus and Fusarium oxysporum.
5.
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