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REVIEW ARTICLE
Isolation of fungi from various habitats and
their possible bioremediation
Farazimah Yakop, Hussein Taha and Pooja Shivanand*
Environmental and Life Sciences Programme, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link BE 1410, Brunei Darussalam
7–9 10
Fungi are the most diverse and adaptable group of liv- pigment and drug production , food industry and
11,12
ing organisms. The literature highlights that coloniza- bioremediation . In this article, the diversity of these
tion density and fungal diversity can be affected by fungi and their isolation from various habitats as well as
factors such as salinity, pH, temperature, altitude and their bioremediation capabilities are discussed.
availability of nutrients. The present article discusses
isolation and characterization of fungi from different Isolation and identification of fungi
ecosystems like forests, mangroves and coastal areas.
Fungal interaction with ecosystems is reported to play Isolation of mycorrhizal fungi involves two methods.
an important role in the biodiversity, survivability, First, soil samples from the rhizosphere of the host plants
propagation and productivity of plants. Fungi have are suspended in water and sieved through meshes of var-
established a beneficial association with numerous ious size. Fungal infection of roots was observed under a
hosts and have the capability to degrade environmen- microscope, and the soil suspension is examined for
tal pollutants such as hydrocarbons and by-products. 13–15
Hence, fungi and fungal products like extracellular spores . In the second method, soil samples were
hydrolytic and lignin-degrading enzymes, and surfac- serially diluted with sterile distilled water at various con-
–1 –7
tants offer eco-friendly and cost-effective strategies to centrations (10 to 10 ) to be used as inocula for the iso-
address pollution, especially with respect to bioreme- lation of fungi. They can be isolated using different
diation of oil spills and polyaromatic hydrocarbons. growth media such as mycological broth agar, potato
dextrose agar, Sabouraud dextrose agar, Czapek’s dox
Keywords: Bioremediation, fungi, habitats, hydrocar- agar and malt extract agar2,4,5. Most studies prefer potato
bon degradation. dextrose agar medium to isolate fungi from environmen-
6,16–18
tal samples .
FUNGI are diverse organisms consisting of seven recogni- Isolation of endophytic fungi is slightly different com-
zed phyla: Ascomycota, Basidiomycota, Blastocladiomy- pared to mycorrhizal fungi as fungal endophytes can only
cota, Chytridiomycota, Glomeromycota, Microsporidia be found within the plant tissues. The two most common
1 methods are scraping method and fragment plating method.
and Neocallimastigomycota . They are ubiquitous, occur- In the scraping method, the sterilized plant material is cut
ring in various types of habitats. Since 1860, many fungi half longitudinally and the pith is scraped with a sterile
have been isolated from various habitats: grasslands, scalpel. The presence of hyphae is determined under a
tropical forests, polar regions, deserts and coastal areas. microscope. This method is limited only to mature plants.
Fungi are considered as one of the most adaptable groups However, in fragment plating method, sections of steri-
of organisms and also as essential soil microbial constitu-
ents2,3. They are essential components of the soil, contri- lized plant materials are placed on prepared media plates
buting to nutrient availability and supporting the and incubated until fungal growth is observed. Later, the
propagation of ecosystems such as mangroves. They are tips of the fungal hyphae are removed and placed in my-
19–24
decomposers of organic matter such as wood, stem and cological media .
leaf, and also function as plant symbionts in ecosystems, Mycological media should contain an antibiotic (such
playing an important role in ecological and biogeochemi- as streptomycin or chloramphenicol) to suppress bacterial
cal processes. Thus, fungi contribute to the degradation of growth. Cultures are incubated at 25–30°C at different
1,2,4,5 time periods, depending on the rate of fungal growth.
organic matter in the ecosystems . The frequency and Fungal colonies with morphological differences are sub-
diversity of fungi vary from place to place, depending on cultured on fresh agar plates to ensure the isolation of
the physical, chemical and biological factors of their 2,4,17,25,26
habitat6. pure cultures .
Apart from playing a key role in the ecosystems, fungi Initially, fungal isolates acquired from the samples are
are also beneficial for industrial and other uses such as identified based on morphology and microscopic exami-
nation. Morphological characteristics could be identified
by the colour and septation of spores, pigmentation and
*For correspondence. (e-mail: pooja.shivanand@ubd.edu.bn) the fruiting body with reference to identification keys.
CURRENT SCIENCE, VOL. 116, NO. 5, 10 MARCH 2019 733
REVIEW ARTICLE
Monographs used to identify the morphology of fungi are Isolation of fungi from mangroves
Standard Manuals of Marine Mycology, the Higher
Fungi, Compendium of Soil Fungi and Genera of Hypho- According to Behera et al.2, mangrove forests are bio-
mycetes2,25,27. diversity hotspots for marine fungi. Mangroves signify
The micro-features of pure culture can be identified by intermediate vegetation sandwiched by land and sea,
semi-permanent microscopic preparations of fungi using which is growing in waterlogged soil with lack of oxy-
lactophenol cotton blue stain. A drop of lactophenol cot- gen. The mangroves soil substrata are generally firm to
ton blue stain is placed on a clean slide before transfer- soft mud with aerial roots known as pneumatophores
36
ring a small tuft of fungus with a sterilized inoculating which emerge from substratum to supply air . Soil is
loop. Then the slides are gently heated to release air bub- among the essential habitats for microorganisms such as
bles, if any, on the glass cover and later observed under bacteria, fungi, nematodes, etc. and also a general reser-
6,17,25,28,29 4
the microscope with oil immersion objective . voir for wind-dispersed fungal spore .
13
Identification of fungi can also be done genetically Gupta reported fungal diversity of different mangrove
and is often used to confirm the initial morphological areas with varying salinity. There were 45 fungi isolated
identification. The most common marker used for species representing 5 genera (Scutellospora C. Walker & F. E.
identification and phylogenetic analysis is the ITS (inter- Sanders, Glomus Tul. & C. Tul., Gigaspora Gerd. &
nal transcribed spacer) region. Trappe, Entrophospora R.N. Ames & R.W. Schneid. and
Acaulospora Gerd. & Trappe). Majority of fungi that
Isolation of fungi from forest ecosystems were found in low salinity areas belonged to the genus
Glomus, and the presence of two Glomus species
Different species of soil fungi are found in forest areas [Glomus intraradices N.C. Schenck & G.S. Sm. and G.
across the world. The most common fungi in forest soils geosporum (T.H. Nicolson & Gerd.) C. Walker] in high-
are Aspergillus Micheli, Penicillium Link, Trichoderma salinity areas suggests the possibility of habitat adapta-
13 37
Pers. and Fusarium Link. They have been found in vari- tion to salt tolerance . Li et al. studied the endophytic
ous types of forest soils such as yellow–brown forest soil, communities in four different mangroves species in China
raw calcareous soil, yellow soil, red soil, mountain– and found that overall colonization frequency on the four
30
meadow soil and brown forest soil , as well as from dark host trees varied from 24% to 33%. Colonization was
brown forest soil, brown coniferous forest soil, mountain also seen to be higher in twigs compared to leaves. Three
31
grass forest soil and mountain tundra soil . hundred and one fungal isolates were obtained from vari-
Several studies have discussed the isolation and cha- ous samples and ITS sequencing indicated that Phomop-
racterization of various strains of fungi from different sis Sacc. & Roum., Phyllosticta Pers., Xylaria Hill ex
forests around the world. Isolation of Trichoderma, Fusa- Schrank, Leptosphaerulina McAlpine and Pestalotiopsis
rium, Acremonium Link, Penicillium and Paecilomyces Steyaert were dominant in all host species.
Samson from Brazilian Savannah and Atlantic rainforest The fungal colonization in grey mangroves, Avicennia
32
has been reported . In Nanga Merit Forest, Sarawak, marina (Forssk.) Vierh. of Red Sea, Saudi Arabia has
Malaysia, isolates of Penicillium and Streptomyces been studied. Six samples were collected and 145 species
33
Waksman & Henrici have been characterized . Paecilo- were identified. Ascomycota was dominant (76–85%) in
myces marquandii (Massee) S. Hughes, Aspergillus fla- all samples followed by Basidiomycota (14–24%). The
vus Link, A. fumigatus Fresenius, Mucor hiemalis prevalence of Ascomycota in mangroves areas is
Wehmer, Penicillium sp., Cladosporium herbarum (Pers.) expected as their lignocelluloses cleaving capabilities are
Link, Trichosporiella cerebriformis (G.A. de Vries & important in areas that are rich in lignin and cellulose,
Kleine-Natrop) W. Gams, Mortierella sp. Coem., Synce- and these fungi would allow the substrates to enter the
phalastrum recemosum Cohn and Cunninghamella echi- food web. Aspergillus and Schizosaccharomyces Lindner
1
nulata (Thaxt.) Thaxt. ex Blakeslee were identified from were found to dominate the soil areas .
Bhadra Wildlife Sanctuary in the Western Ghats, India,
comprised of dry and moist deciduous, semi-evergreen
34 Isolation of fungi from coastal areas
and evergreen forests . Another study carried out in the
35
same area by Banakar and Thippeswamy revealed other
fungal species, such as Chaetomium globosum Kunze, Fungi are known to exist in marine environments since
38
Thelephora terrestris Ehrh., Humicola sp. Traaen, Tala- the early times . The ocean reflects a great reservoir of
romyces sp. C.R. Benj., Trametes versicolor (L.) Lloyd, biodiversity. The marine environment is exceptionally
Trametes hirsuta (Wulfen) Lloyd, Phanerochaete sordida complex and comprises a broad variety of fungal diver-
39,40
(P. Karst.) J. Erikss. & Ryvarden, Lenzites betulina (L.) sity . Marine fungi are a taxonomically diverse group,
Fr., Pleurotus ostreatus (Jacq.) P. Kumm., Stereum ost- developing in many different habitats within the marine
rea (Blume & T. Nees) Fr. and Phanerochaete chrysos- ecosystems, including sea water, corals, algae, marine
41,42
porium Burds. sediments, etc. . These marine fungi can be sub-divided
734 CURRENT SCIENCE, VOL. 116, NO. 5, 10 MARCH 2019
REVIEW ARTICLE
into two groups: (1) obligate or residents, and (2) faculta- plant–fungus relationships are ectomycorrhizal (ECM)
tive or transient fungi. Obligate marine fungi grow and and arbuscular mycorrhizal (AM). ECM is characterized
sporulate exclusively in marine or estuarine environments by the formation of a fungal coating on the root surfaces
whereas facultative marine fungi inhabit terrestrial or and of hyphal network penetrating between the cortical
50
freshwater habitats, but are capable of growing and spo- cells and endodermis, known as Hartig net . This type is
41
rulating in marine environments . Marine-derived fungi usually associated with coniferous and deciduous trees.
have been found in several places such as sediment, However, AM is typically associated with herbaceous
43
sponges, algae, fish, deep sea and mangrove wood . The plants and grasses, and is characterized by the formation
51,52
interest in marine ecology has also increased worldwide. of arbuscules within the root cortical cells .
Several substrata in marine environments are good These mycorrhizal symbioses enhance plant growth by
38
sources for the detection of fungi . Marine fungi have allowing the soil to be more efficiently utilized, resulting
numerous ecological roles such as degradation of biota, in more uptake of nutrients such as nitrogen and phospho-
provision of chemical protection, pathogenicity, symbi- rus, as well as increasing the plant defence mechanism
osis and impact on various holobiont groups42. Some ma- against natural stresses. Nitrogen plays an important part
rine fungi are key decomposers of organic matter, and in plant morphology, photosynthesis, formation of nucle-
41
others are involved in the denitrification process and ic acids and enzymes, whereas phosphorus is needed for
44 51
hence are considered as a source of industrial enzymes . cell division, reproduction and metabolism . In return,
45
Beena et al. carried out a study on the diversity of the fungi benefit from these photosynthesizing plants as
fungi from the coastal sand dunes in India, and isolated the fungi have direct access to the carbohydrates translo-
38 species from 5 genera (Scutellospora, Glomus, Gigas- cated from the leaves into the roots.
pora, Sclerocystis Berk. & Broome and Acaulospora) The mycoheterotrophic relationship of three common
with Glomus mosseae (T.H. Nicolson & Gerd.) Gerd. & shrubs of the family Pyroleae found on the northern side
15
Trappe being the most dominant. Séne et al. studied the of Mt Fuji, Japan, with ECM fungi was studied. Pyrola
diversity of ectomycorrhizal fungi in Coccoloba uvifera alpina H. Andres, P. incarnata (DC.) Freyn and Orthilia
(L.) L. mature trees, and found 8 species colonizing the secunda L. were found to have shared similar mycobionts
sporocarps and sclerotia whereas 15 species were found with the surrounding trees, Larix kaempferi (Lamb.)
on the root tips, with the seedlings sharing 14 of these Carr., Abies veitchii Lindley and Betula ermanii Cham,
taxa. Scleroderma bermudense Coker, Russula cremeoli- but at different levels. P. incarnata shared almost 70%
lacina Pegler and Thelephoraceae were dominant. S. mycobionts with the surrounding trees, whereas P. alpina
bermudense had the potential to form a common and O. secunda shared 32% and 15% respectively. O. se-
mycorrhizal network between the mature trees and cunda was exclusively associated with Wilcoxina sp.
seedlings, for increasing the survival chances of the Chin S. Yang & Korf, an ECM fungus, suggesting an
seedlings. independent mycorrhizal association. A more diverse
46
Parveen et al. explored the fungal diversity in Maha- community of ECM fungi was found in the other two
nadi River, India. There were 31 fungal species isolated, Pyrola species, with a higher percentage of shared myco-
53
with Aspergillus niger Tiegh. being the most prominent. bionts .
Another study in 2016 had isolated 8 fungal genera (As- Some orchid plants from the forests in Thailand, par-
pergillus, Penicillium, Thielavia Zopf, Fusarium, Emeri- ticularly Aphyllorchis montana Rchb. f, A. caudate Rolfe
cella Berk., Cladosporium Link, Scytalidium Pesante and ex Downie and Cephalanthera exigua Seidenf. were
47
Alternaria Nees) from Masturah, Saudi Arabia . Most found to have mycoheterotrophic association with ECM
isolated fungi showed significant growth on petroleum fungi. A. montana and A. caudata showed a diverse ECM
media and hence have the potential for biodegrading community, majority of which were Russulaceae and
crude oil-based substances. Since 1957, the Japanese An- Thelephoraceae. The latter was also found predominantly
tarctic Research Expedition has isolated 76 fungal species in C. exigua (65%), but the same orchid species did not
from the Syowa Station, East Antarctica. Majority of show high ECM diversity as in the other two species,
54
these fungi were Ascomycota and Basidiomycota. They which could be due to its short roots .
55
were able to survive in harsh conditions such as low tem- de Souza and Freitas compared AM fungi of an exotic
48
peratures and lack of nutrients . invasive plant, Prosopis juliflora (Sw.) DC. and a native
plant, Mimosa tenuiflora (Willd.) Poir. that grow in the
Isolation of mycorrhizal fungi tropical seasonal dry forest in Brazil. In both plants, sig-
nificant differences were observed in fungal composition
as well as in the soil chemical factors such as soil pH,
Fungi that form localized hyphae interfaces have syn- total organic carbon, total nitrogen and availability of
chronized development with the host plants and benefit phosphorus. A total of 29 species of AM fungi were
the host plants via nutrient transfer are known as mycorr- found in all of the soil samples with the orders Glome-
49
hizae . The most common types of these symbiotic rales (44.8%) and Gigasporales (41.4%) and the genus
CURRENT SCIENCE, VOL. 116, NO. 5, 10 MARCH 2019 735
REVIEW ARTICLE
Funneliformis C. Walker & A. Schüßler being the most stress had reduced the growth of Pinus muricata D. Don.
prevalent. The soils where P. juliflora developed had A total of 82 species were isolated, and preferential root
higher chemical factors, but only consisted of AM fungi colonizers such as Suillus tomentosus (Kauffman) Singer
from the orders Diversisporales, Gigasporales and Glo- and Russula brunneola Burl. were found on pygmy and
merales. The soils where M. tenuiflora developed also nonpygmy host plants respectively. It was also found that
had AM fungi from the same orders but also with the the trees from the more stressful environment were pre-
order Archaeosporales, making the AM community more ferentially colonized by ECM fungi with more carbon-
diverse. intense foraging strategies62.
Swietenia macrophylla King seedlings and mature trees
growing in a tropical rainforest in Mexico, were studied Isolation of endophytic fungi
for their AM fungal communities. Almost 23 fungi were
isolated and classified into 4 genera. The diversity of AM
fungi in the mature trees composed of Glomus (52.3%), Endophytic fungi live within plant tissues, such as leaves,
Acaulospora (38%), Ambispora C. Walker, Vestberg & twigs, branches and roots63. These fungi produce metabo-
A. Schüssler and Gigaspora (4.7% each). Acaulospora lites that generally cause no harm to the plants, but can
63,64
(63.6%) and Glomus (36.3%) were isolated from the increase the survival fitness of the hosts . Differences
56 57
seedlings . Lang et al. studied the diversity of mycorr- in the species composition of fungal endophytes can be
hizal species in mixed deciduous forests in Germany. A observed in trees with different growth rates, as shown in
total of 130 ECM fungal species were detected on the a study where 43 fungi were identified from 360 twigs of
65
root tips of host plants (Fagus sp. L., Tilia sp. L. and Pinus sylvestris L. (Scots pine) in northern Spain . The
Carpinus sp. L.). These species colonized 92.8% of the most abundant fungi were from the classes Dothideomy-
root tips, and AM fungi were abundant in the roots of cetes and Sordariomycetes. The endophytic community
Fraxinus sp. L. and Acer sp. L. between fast- and slow-growing pines was found to be
Souza and Rodrigues studied the diversity of AM fungi different, with Phoma herbarum Westend. occupying the
in three mangrove species (Acanthus ilicifolius L., Ex- twigs of fast-growing trees while Hypocrea lixii Pat. was
65
coecaria agallocha L. and Rhizophora mucronata Lam.) associated with the slow-growing trees .
from two mangrove forests in India and during different Rojas-Jimenez et al.66 explored the diversity of endo-
seasons (pre-monsoon, monsoon and post-monsoon). phytic fungi along an altitudinal gradient (400–2900 m)
A total of 11 AM fungi from five genera (Rhizophagus in tropical forests of Costa Rica. They found that the
P.A. Dang., Glomus, Funneliformis, Racocetra Oehl, lower the altitude, the higher was the species richness of
F.A. Souza & Sieverd. and Acaulospora) were isolated endophytic fungi; also particular preference to plant host
from all sites and during all seasons. Species richness and for fungi in higher altitudes was uncommon. Almost 92%
spore density were recorded highest in pre-monsoon sea- of these isolates were from the class Sordariomycetes.
son and lowest in monsoon season. The high spore densi- Colletotrichum Corda, Diaporthe Nitschke and Xylaria
ty in pre-monsoon season could be explained by soil were dominant at different altitudes since many years.
temperature, as higher temperature favours AM fungal The diversity of fungal endophytes in Fortunearia
58
sporulation, or by other factors . Studies of ECM fungi sinensis Rehder & E.H. Wilson growing in mixed ever-
from mangroves are limited as the fungal communities in green/deciduous broadleaved forest in Jiangsu Province,
59 67
mangroves are typically AM . China was studied . Almost 1436 strains were isolated
In a study, coastal sands and reclamation sites in Icel- from the laminae, petioles and twigs of F. sinensis that
60
and were examined for AM fungal abundance . On the were collected during spring and autumn. These isolates
coastal sand plain, AM fungi were not found on the bar- were classified into 33 genera with Alternaria (21.52%),
ren sand, while some were discovered in a natural old Fusarium (19.64%) and Pestalotiopsis (13.16%) being
dune system and in the reclamation sites of Leymus are- the dominant ones. The genus Alternaria was commonly
narius (L.) Hochst. However, the older reclamation site found in both seasons. However, Pestalotiopsis and
had a higher number of AM fungi compared to new sites. Chaetomium Kunze were more abundant in spring, while
The same was observed on the volcanic island of Surtsey. Fusarium and Monotospora Corda in autumn. The diver-
Different AM inocula of L. arenarius revealed that indi- sity of endophytic fungi in F. sinensis was higher in the
genous AM fungi were able to improve the growth of petioles than laminae and twigs67.
60
seedlings . A study was carried out on a New Zealand In the Western Ghats, Pestalotiopsis spp. were isolated
dune, where root samples of Spinifex sericeus R.Br. were from 58 tree species from 4 types of forests (dry thorn,
collected61. A total of 22 AM fungal operational taxonomic dry deciduous, stunted montane evergreen and moist
units (OTUs) were identified and classified into 7 genera; deciduous). The frequency varied from 0.7% to 41.0% with
the dominant genera were Rhizophagus and Racocetra. regard to forest type and tree host. ITS sequencing deter-
The association of ECM fungi and trees in relation to mined that 28 of these isolates, which were identified (on
environmental stress was examined, where environmental the basis of morphology) as Pestalotiopsis, were discovered
736 CURRENT SCIENCE, VOL. 116, NO. 5, 10 MARCH 2019
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