NewBioWorld A
Journal of Alumni Association of Biotechnology (2020) 2(1):18-24
RESEARCH
ARTICLE
Leaf Surface
Mycoflora around the Hospital Area with Special Reference to Dr. Bhim Rao
Ambedkar Memorial Hospital, Raipur, Chhattisgarh
B.M. Lall
Department
of Botany, Govt. D. B. Girls P.G. (Auto.) College, Raipur, Chhattisgarh, India
lallpinky@yahoo.co.in
Corresponding Author Email- lallpinky@yahoo.co.in
ARTICLE INFORMATION
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ABSTRACT
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Article history:
Received
23 October 2019
Received in revised form
28 December 2019
Accepted
Keywords:
Leaf surface;
Mycoflora;
Hospital;
Aerosol
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Leaf surface provide an important substrate for the
growth of a wide variety of fungal organisms. Saprophytic fungi obtain water
and nutrients from these surface and leaf surface provide a physical
environment suitable for growth and reproduction. For phytopathogenic fungi,
the leaf surface represents a temporary, supportive environment, but a
surface must be breached before a successful pathogenic interaction can
become established. Thus, the physical and chemical characteristics of the
leaf surface play an important role in governing the success or failure of
fungal growth on, and subsequently in, the leaf. The present paper deals with
leaf surface mycoflora around the
hospital area with special reference to Dr.Bhim Rao Ambedkar Memorial Hospital,
Raipur Chhattisgarh was done by using leaf
suspension on PDA (Potato
Dextrose Agar) medium for the period of one year from July 2006 to June 2007.
During the present study, total 46 fungal species (173 colonies) belonging to
22 genera were recorded. Class wise percentage contribution were recorded and
showed that, Anamorphic fungi contributed maximum (96.53%) followed by Zygomycotina
(2.87%) and Ascomycotina (0.58%). Season wise total percentage contribution of
leaf surface mycoflora were also recorded and showed that maximum
contribution were observed in winter season (48.55 %), moderate (30.64%) in
rainy season and minimum (20.81%) in summer season. Similarly, maximum contribution were found
in the month of November (24.86%) and minimum contribution were observed
during the month of June (3.47%). It is also shows that fungal species i.e. Cladosporium
sphaerospermum (12.13%), C. Cladosporioides (8.09%), Alternaria alternata (7.51%),
Aspergillus niger and Curvularia lunata (5.75%), Phoma fimeti (4.62%) and Aspergillus
flavus (4.04%) were most contributed leaf surface mycoflora. It is also
observed that, Cladosporium sphaerospermum (23.80%) and C. Cladosporioides (10.71%)
were most dominated in winter season, while Alternaria alternata and Aspergillus
niger (13.20%) were dominated in rainy season. The presence of these fungi supports the idea that the
air spora constitutes the source of many fungi that can potentially colonize
the leaf surface.
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Introduction
Aerobiology
deals in large parts with bio particles present in air. Some of fungal flora
may infect the plants of the standing crop in the field and as a result
spreading of diseases occurs. Mycoflora of leaf surface (i.e. Phylloplane)
varies in size and diversity depending on the influence of numerous biotic and
abiotic factors which affect their growth and survival (Bakkar et al., 2002). Leaf
surface is the most suitable platform for airborne microorganisms. On
availability of suitable microhabitat, these fungal spores get settled down on
this platform and try to colonies on it. Finally a triangular relationship is
developed among leaf surface, fungal spores and the environment (Tilak et al.,
1981).
DOI: 10.52228/NBW-JAAB.2020-2-1-4
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The phylloplane, the surface of plant leaves, is a complex terrestrial
habitat that is characterized by a variety of microorganisms including
bacteria, filamentous fungi and yeast. Pathogens, saprobes and epiphytes occur
in this habitat and numerous studies have described the phylloplane populations
from various plant species (Breeze and Dix, 1981; Mishra and Dickinson, 1981;
de Jager et al., 2001; Andrews et al., 2002; Ina´cio et al., 2002; Osono et
al., 2004). The non-pathogenic fungi that inhabit the phyllosphere depend on
nutrients exuded from the leaf or those deposited from the atmosphere (Belanger
and Avis, 2002; Ina´cio et al., 2002). In addition to nutrient levels, growth
and abundance of phylloplane fungi are also influenced by environmental
conditions such as water availability, UV radiation, and temperature (Breeze
and Dix, 1981; Newsham et al., 1997; Sundin, 2002; Zak, 2002).
The air
spora constitutes both the source of fungi that colonize the leaf surface and
the sink of spores released from the leaf surface by various dispersal
mechanisms (Pedgley, 1991; Kinkel, 1997; Aylor, 2002). Airborne spores impact
on leaf surfaces and may adhere due to structural or chemical features of the epidermis
and the spore (Andrews and Buck, 2002). Spore release from many fungi
inhabiting the phylloplane is passive through the action of wind or rain
splash; however, other spores are actively propelled into the atmosphere by
various mechanisms (Kinkel, 1997; Aylor, 2002; Levetin, 2002).
Leaf
surface provide an important substrate for the growth of a wide variety of
fungal organisms. Saprophytic fungi obtain water and nutrients from these
surface and leaf surface provide a physical environment suitable for growth and
reproduction. For phytopathogenic fungi, the leaf surface represents a
temporary, supportive environment, but a surface must be breached before a
successful pathogenic interaction can become established. Thus, the physical
and chemical characteristics of the leaf surface play an important role in
governing the success or failure of fungal growth on, and subsequently in, the
leaf. Numerous reviews have been written about the growth of fungi in
association with plant surfaces (e.g., Preece, 1976; Aist, 1981; Wynn, 1981;
Staples and Macko, 1984; Hoch and Staples, 1987, 1991).
The
present study was undertaken to study leaf surface mycoflora around the
hospital area with special reference to Dr. Bhim Rao Ambedkar Memorial
Hospital, Raipur, Chhattisgarh to find that some leaf surface fungi are major
contributors to the air spora.
Materials and Methods
Study area
Raipur District
is the capital of Chhattisgarh; it is situated in the fertile plains of
Chhattisgarh region. This District is
situated between 22º - 33’ North Latitude and 82º - 38’ East Longitude about
298.60 meters above the sea level. The District is surrounded by District
Bilaspur in North, District Baster and part of Orissa State in South, District
Raigarh and part of Orissa State in East and District Durg in West. The climate
of Raipur city is characterized by the rainy season (July-October), winter
season (November-February) and summer season (March-June). Dr. Bhim Rao
Ambedkar Memorial Hospital, Raipur is the largest government health care
facility in Chhattisgarh. This is located in the heart of the city and within
one kilometer from railway station and bus stand. Dr. Bhim Rao Ambedkar
Hospital is commonly known as MECAHARA (Medical College Hospital Raipur).
Sample Collection
For the
isolation of leaf surface mycoflora, leaves were collected from different
plants which are presented in the surrounding areas of the hospital at
fortnightly intervals at all the time (July, 2006 to June, 2007). Different
leaves of plants (available at outside of the hospital area) are collected with
the help of sterilized forceps in sterilized polythene bags.
Isolation of fungi
After that
leaves are brought into the laboratory for the isolation of leaf surface
mycoflora. In the laboratory, the collected leaves are placed in 150 ml of
conical flask containing 75 ml of sterilized distilled water. The flasks are
hand shaken for 30 minutes to make a homogenous suspension of microorganisms
attached to the leaf surface. This suspension was used for the isolation of
leaf surface mycoflora. 0.1 ml of this suspension is poured into the each
petriplates (triplicate PDA plates) with the help of micropipette. After that
petriplates are incubated at 26 ± 1º C for incubation period. (Tiwari, 1977).
Media Preparation Composition of
Potato Dextrose Agar Medium:
Potato (peeled) - 250gm
Dextrose - 20 gm
Agar - 15 gm
Distilled water - 1000 ml
Preparation of Potato Dextrose Agar
Medium:
The potato
tubers were peeled and weighed for about 250g. The tubers were chopped into
small pieces with the help of sterile knife. The chopped potatoes were
transferred into a conical flask containing about 1000ml of distilled water.
The content was boiled for 20 min. The supernatant were decanted and filtered
by muslin cloth and the filtrate was collected. Dextrose (20g) and agar (15g)
were transferred into the extract and shaked to dissolve the ingredients.
The medium
was made up to 1 litre by addition of distilled water. The pH of the medium was
adjusted to 5.6. Finally, the medium was cotton plugged and autoclaved at 121ºC
for 15 minutes.
Lacto Phenol Cotton Blue Mounting:
A portion
of the mycelium of the representative colonies was picked up with the help of a
pair of needles and semi-permanent slides were prepared using lactophenol
cotton blue (20g – phenol (crystal); 20g lactic acid; 40g glycerin; 20 ml
water; cotton blue a few drops). The slide was gently heated in a sprit lamp so
as to release the air bubbles, if any present inside the cover glass. The
excess stain was removed using tissue paper and the cover glass was sealed
using white nail polish.
Identification of fungi:
As the
plating method yield facultative phylloplane fungi were identified referring
the standard manuals. The manual of soil fungi (Gillman, 1857) and Dematiaceous
Hyphomycetes and More Dematiaceous Hyphomycetes (Ellis, 1976). Cultures were
incubated at room temperature for 5–7 days; colonies were counted and then
examined microscopically. Standard keys were used for colony identification
(Ellis, 1971, 1976; Barnett and Hunter, 1998)
Ecological studies
For
ecological studies, at the end of the incubation period, percentage
contribution of the fungal flora is calculated (Prasad and Bilgrami, 1969;
Jadhav and Tiwari, 1994) with the help of the following formula:-
Results and Discussion
During the
present investigation, total 46 fungal species (173 fungal colonies) belonging to 22 genera were observed (Fig-1).
02 fungal species i.e. Mucor sp. and Cyncephalastrum recemosum (05 fungal
colonies) were captured from Zygomycotina and only single species i.e. Lewia infectoria (01 fungal colony) was
investigated from Ascomycotina. 43 fungal species (167 fungal colonies) were
isolated from Anamorphic fungi.
Climatic
factor such as temperature, relative humidity and rainfall have been known to
play a major role in the concentration of the fungal population of a particular
area. During the present investigation, maximum 33 fungal species (84 fungal
colonies) are observed in the winter season due to favorable temperature and
relative humidity (28.9 ºC and 82.1% respectively), which are favorable for the
fungal growth. Moderate 27 fungal species (53 fungal colonies) are observed in
rainy season due to moderate temperature (30.7 ºC) and relative humidity (91.1%)
and due to the washing off, of the fungal spores by the rains clearing the
atmosphere. While, minimum 24 fungal species (36 fungal colonies) were recorded
during summer season due to unfavorable temperature (37.8 ºC) and relative
humidity (64.2%). Very high temperature during summer season is unfavorable for
the sporulation of fungal spores. These studies are in agreement with the
findings of several other workers in Raipur (Tiwari and Sahu; 1988; Tiwari and
Sahu, 1989; Sahu1996; Sahu 1998; Sharma, 2001; Saluja, 2006; Singh, 2006 &
Tiwari and Khare, 2012). Tilak (1982) also find the lower incidence
of mycoflora during summer may be attributed to unfavorable climatic condition.
Maximum number of fungal spores during winter season has been confirmed by a
number of aerobiologist (Cunningham, 1873; Rajan et. al., 1952; Hirst, 1953; Sreelamulu and Ramalingam, 1966; Nagarjan et.
al., 1976; Tiwari 1977; Tiwari and sahu, 1989 and Nayak et. al., 1998). Similarly Maximum
incidence of fungal species and colonies were recorded in November, July and
December (24.86%, 11.56% and 9.83% respectively). While minimum number was
recorded in the month of June (3.47%) (Fig- 2 & 3).
In our
findings Zygomycotina was present in rainy and winter season and absent in
summer season. Ascomycotina was present only in winter season and absent in
Rainy and summer season. Maximum number of fungal species and fungal colonies
were encountered in the months of October, November and December due to the
favorable environmental condition, while minimum in the month of September and
mostly in summer season due to high temperature and minimum relative humidity.
Cladosporium sphaerospermum (12.13%) most contributed
leaf surface mycoflora followed by Cladosporium
cladosporioides (8.09%), Alternaria
alternata (7.51%), Aspergillus niger,
Curvularia lunata (5.75%), Phoma
fimeti (4.62%) and Aspergillus flavus
(4.04%). In rainy season Alternaria
alternata, Aspergillus niger (13.20%) and Phoma fimeti (9.43%) were most dominated. In winter season Cladosporium sphaerospermum (23.80%) contributed maximum followed by Cladosporium cladosporioides (10.71%)
and Alternaria alternata (7.51%).
Similarly in summer season Aspergillus
flavus, Cladosporium cladosporioides, Curvularia lunata and Pestalotiopsis glandicola (8.33%) most
contributed fungal species on leaf surface of the plants. Leaf surface fungi
identified also parallel those found in other studies (Breeze and Dix, 1981; Mishra and
Dickinson, 1981; Tiwari and Sahu, 1989; Sahu,1992, 1996; McCormack et al.,
1994; Andrews et al., 2002; Ina´cio et al., 2002; Saluja, 2005; Singh, 2006; Tiwari
and Khare, 2012;). The presence of these fungi supports the idea that the air
spora constitutes the source of many fungi that can potentially colonize the
leaf surface (Pedgley, 1991; Kinkel, 1997; Aylor 2002). In our findings Alternaria alternata (25%) prominent in
July, Aspergillus niger (44.66%) in August, Cladosporium sphaerospermum (46.51%) in
November, Alternaria citri (11.76%)
in December, Cladosporium
clamydospora, Aspergillus flavus, and
A. chevalieri var. intermedius (20%) in May and Curvularia clavata ( 33.33%) were dominated in the month of June. The dominance of Alternaria, Aspergillus
and Cladosporium is in agreement
with observation of several scientists (Agrawal et. al.,1969; Collins et.
al., 1973; Rati and Ramalingam 1976, Janaki Bai and Subba Reddi,1981;
Shastri, 1981; Sahney and Purwar, 2002; Saluja,
2005; Singh, 2006; Abdel-Hameed et. al., 2007; Sabariego et. al. 2007
and Tiwari and Khare, 2012).
Class wise
percentage contribution showed that Anamorphic fungi contributed maximum
(96.53%) followed by Zygomycotina (2.89%) and Ascomycotina (0.58%) (Fig-4). The
percentage contribution of fungal colonies of Anamorphic fungi are maximum
throughout the study periods. Maximum percentage contribution of Anamorphic
fungi are also reported by several workers (Verma and Khare, 1987; Manoharachary
et. al., 1988; Satheesh and Rao, 1994; Sharma, 2001; Saluja,
2005; Singh, 2006 and Zoppas et al.
2006). Winter season contributed maximum number of
fungal flora (48.55%) followed by rainy season (30.64%) and summer season
(20.81%) (fig-5). Similarly, maximum numbers of fungal colonies were
encountered in the month of November and July (24.86% & 11.56%)
respectively. while minimum in the month of June (3.47%) (Fig- 6).
Conclusion
The phylloplane, the surface of
plant leaves is a complex terrestrial habitat that is characterized by a
variety of microorganisms including bacteria, filamentous fungi and yeast.
Phylloplane fungi are the mycota growing or the surface of leaves. There are
two groups of phylloplane fungi: residents and casuals. Residents can multiply
on the surface of healthy leaves without noticeably affecting the host.
Whereas, casuals land on the leaf surface but cannot grow.
Microorganism are introduced into air from
various sources, the chief source of these microorganism are soil and
vegetation of particular area. Microorganism which are found on the surface of
plants either as pathogens or as saprophytes also get suspended in air. Man
also contributes to this to a great extent by disturbing the atmosphere. Leaf surface mycoflora have been studied in
this connection and surface of the leaves have been found to be responsible for
the air microflora, the most common sources of air microflora are soil, leaf
and water of corresponding areas ( Lall, 2008). Microorganisms of the particular area are the
important source of the airspora of that particular area. Different types of
microorganisms present in the soil and different type of microorganisms present
in the plants or its parts and different type of the microorganisms, which are
present on the dead organic matter, are the important source of the airspora of
the particular area. Fungal spores can be serious problem for human health.
Some fungal spores play a significant role in plant pathology and human
respiratory allergy. The allergenic nature of Alternaria, Aspergillus, Cladosporium, Curvularia and Penicillium has already been established
(Gupta, et al., 1960; Al-Doory et al., 1982; Singh et al., 1994; Kurkela, 1997;
Corden and Millington, 2001). Monitoring of leaf surface fungi can be helpful
in prevention of plant diseases and fungal allergic diseases.
Sustainable
agriculture always emphasizes on preserving the soil quality and enhancing the
crop yield. It also focuses on the well-functioning of soil ecosystem. However,
plants have to face multiple environmental challenges (variety of stresses)
everyday which affects its overall physiology, growth and development thereby
reducing the yield and productivity. The production of reactive oxygen species,
disturbing the plant nutrient homeostasis, affecting photosynthetic processes
and other metabolic cycles are the common outcomes of any stress. Heavy metal
contamination is becoming one of the important stressor against which the plant
should initiate the defensive mechanism. And the application of biostimulants
is one such support to enhance the defensive approach to regulate and control
the overall processes of growth and development. Biostimulants fight various
biotic and abiotic stresses through a combination of an array of mechanisms.
Thus, the purpose of this review is to briefly explain the biostimulant, its
types and the role of few important biostimulant to ameliorate the toxic effect
of heavy metals. This will provide a basis to understand the types of
biostimulants and open the area to study their detail mechanism in response to
heavy metal toxicity. The overall impact of this review will be to set a
scientific frame to identify how the plant biostimulants treatments (substances
and/or microorganisms) have the potential to enhance plant resilience to
nutrient limitation typical of organic farming, and consequently reducing the
gap between organic and conventional yields.
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