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\date{}

\begin{document}

\textbf{Facets of plants of phyllosphere arena of Green Belt Area in
response to seasonal variations on the fungal Biodiversity}

\textbf{Yusra Mariam\textsuperscript{*1} and Ragini
Gothalwal\textsuperscript{2 }}

\textbf{\textsuperscript{1,2} Department of Biotechnology, Barkatullah
University, Bhopal-462026 (M.P.), India}

\textbf{*Corresponding Author:
\href{mailto:mariam29yusra@gmail.com}{\nolinkurl{mariam29yusra@gmail.com}}}

\textbf{ORCID ID-0009-0001-9963-1421}

\textbf{ABSTRACT} :

\textbf{Background:} The phyllosphere, represents a dynamic and hostile
microbial habitat directly exposed to fluctuating macro-environmental
conditions. In urban ecosystems, green belts play a vital role in
mitigating pollution, yet their microclimates and anthropogenic
pressures heavily influence microbial biodiversity.

\textbf{Objective:} This study evaluates the geographical and seasonal
influences on the fungal biodiversity within the phyllosphere arena of
diverse plant hosts across the green belt areas of Bhopal, Madhya
Pradesh, India.

\textbf{Methods:} Leaf samples were systematically collected across
three distinct seasons (Winter, Rainy, and Summer) from key native tree
species, including \emph{Azadirachta indica} (Neem), \emph{Mangifera
indica} (Mango), \emph{Saraca asoka} (Ashoka), \emph{Szygium cumini}
(Jamun) etc. with the help of the Capital Project Administration (CPA)
and SCFRND (Social Conservation of Forest Research and Development) and
Madhya Pradesh Pollution Control Board (MPPCB).Fungal isolates were
cultured, morphologically identified, and analyzed to determine seasonal
distribution and host-plant specificity.

\textbf{Results:} A total of 184 fungal isolates spanning 18 distinct
categories were obtained. Fungal community structures exhibited sharp
seasonal successions driven by temperature and moisture availability.
\emph{Mucor sp.} (28.3\% of total isolates) and \emph{Fusarium sp.}
(19.6\%) strongly dominated during the warm, humid Rainy season,
benefiting from rain-splash dispersal and high relative humidity.
Conversely, \emph{Trichoderma sp.} and \emph{Aspergillus niger}
exhibited peak prevalence during the cooler Winter months, capitalizing
on night-time dewfall. The harsh, arid Summer season (\textgreater40 ◦C)
created a severe population bottleneck, selecting strictly for highly
thermotolerant, heavily pigmented spore-formers, notably
\emph{Aspergillus terreus} and \emph{Aspergillus flavus}.
Geographically, urban green belt plants exposed to vehicular emissions
supported a high density of robust, melanin-rich \emph{Aspergillus}
species. Host-plant architecture also influenced colonization, with
ecological generalists (\emph{Mucor sp.} and \emph{A. niger})
establishing a footprint across a broad spectrum of hosts (e.g., Mango,
Guava, Neem), while genera like \emph{Microsporum} and \emph{Acremonium}
displayed strict specialization toward \emph{Syzygium cumini} (Jamun).

\textbf{Conclusion:} These findings demonstrate that the phyllosphere
arena in Bhopal\textquotesingle s urban green belts is a highly
orchestrated ecosystem where fungal biodiversity is strictly governed by
a tripartite interplay of geographical microclimates, sharp seasonal
transitions, and host-plant identity. This study highlights the
resilience of specific fungal bioindicators in urban forest management.
which implies that different growing seasons should be considered in the
exploitation of the interactions between phyllospheric microorganisms
and host plants.

\textbf{Keywords:} Phyllosphere, Fungal Biodiversity, Green Belt,
Seasonal Variation, Urban Ecology.

\textbf{INTRODUCTION:} Leaves play vital role in supporting
phyllospheric microbes.The phyllosphere, defined as the aerial surfaces
of terrestrial plants, represents one of the largest and most dynamic
microbial habitats on Earth, with a global surface area estimated at
approximately up to 4×10\textsuperscript{8} km\textsuperscript{2} which
is nearly 78.4\% of the total surface area of the earth and is natural
habitat for up to 10\textsuperscript{26} bacterial cells.(Kembel et al.,
2014, Wei et al., 2017). While often perceived as a harsh environment
due to rapid fluctuations in temperature, UV radiation, and nutrient
availability, the phyllosphere supports a vast diversity of
microorganisms, among which fungi play indispensable roles (Vacher et
al., 2016). These epiphytic fungal communities are central to ecosystem
functioning, acting as decomposers of plant exudates, first-line
colonizers in leaf senescence, and protective barriers against
phytopathogens (Arnold et al., 2003). The difference of the microbial
composition of the phyllosphere includes algae, bacteria, filamentous
fungi, and yeast (Morris et al., 2002). Most frequently representing
(44.4\%) of the total isolated fungi species found in the phyllosphere
were \textbf{\emph{Acremonium, Fusarium} sp\emph{, Aspergillus sp,
Geotrichium sp. }}and \textbf{\emph{Penicillium sp.}(}Isiaka, et al.,
2007).

In urban landscapes, "green belt" areas serve as critical ecological
corridors that mitigate the impacts of urbanization, such as the heat
island effect and air pollution (Shukla et al., 2024). However, the
biodiversity within these green belts is increasingly subject to
anthropogenic stressors, including foot traffic and industrial
emissions, which have been shown to significantly alter the alpha and
beta diversity of microbial communities (Perreault \& Laforest-Lapointe,
2022). In a subtropical context like Bhopal, known for its unique
topography and extensive green cover, the interplay between
environmental factors and microbial distribution remains an area of
active investigation.

Previous studies in the Bhopal region have established that fungal
populations---both in the soil and associated with plant roots---exhibit
marked seasonal variation influenced by temperature, rainfall, and
relative humidity (Kaushal and Shukla et al., 2024). Despite this, the
specific geographical and seasonal drivers of fungal biodiversity in the
phyllosphere arena across Bhopal\textquotesingle s urban green belts
have not been fully elucidated. This study aims to characterize the
fungal biodiversity in the phyllosphere of diverse plant species across
selected green belt areas of Bhopal. By examining the influences of
geographical location (site-specific factors) and seasonal transitions
(monsoon, winter, and summer),These external drivers shape the
composition and stability of the foliar mycobiome. Such insights are
vital for assessing the health of urban ecosystems and for developing
strategies for biodiversity conservation.

\textbf{MATERIAL AND METHODS :}

\textbf{2.1 Sampling :}The population is divided into ten sub-groups or
clusters. A simple random sample is taken from each cluster or group.
The ten different sampling sites that are green belt areas from Bhopal
City ,Madhya Pradesh, India were selected according to the survey
conducted with the help of Capital Project Administration (CPA) and
Social Conservator of Forest Research and Development (SCFRND) and
Madhya Pradesh Pollution Control Board (MPPCB)

\includegraphics[width=5.81607in,height=2.91667in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image1.jpg}

\textbf{Fig.1 Map of the study area showing different green belt areas
of Bhopal, (M.P.) India (\url{https://maps.google.com/})}

\textbf{Table 1. Different sampling sites along with codes and list of
plantations in Bhopal City provided by CPA Division-I}

\begin{longtable}[]{@{}
  >{\raggedright\arraybackslash}p{(\columnwidth - 8\tabcolsep) * \real{0.1159}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 8\tabcolsep) * \real{0.3564}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 8\tabcolsep) * \real{0.1349}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 8\tabcolsep) * \real{0.1401}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 8\tabcolsep) * \real{0.2526}}@{}}
\toprule()
\begin{minipage}[b]{\linewidth}\raggedright
\textsubscript{S.No.}
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
\textsubscript{Name of plantation site}
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
\textsubscript{Code}
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
\textsubscript{Year}
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
\textsubscript{Number of Plants}
\end{minipage} \\
\midrule()
\endhead
\textbf{\textsubscript{1.}} & \textbf{\textsubscript{Patel college
Ratibad}} & \textbf{\textsubscript{P.R.}} &
\textbf{\textsubscript{2019-2020}} & \textbf{\textsubscript{2080}} \\
\textbf{\textsubscript{2.}} & \textbf{\textsubscript{Manuabhan Tekri}} &
\textbf{\textsubscript{M.T.}} & \textbf{\textsubscript{2021-2022}} &
\textbf{\textsubscript{410}} \\
\textbf{\textsubscript{3.}} & \textbf{\textsubscript{MANIT-Campus
TYPESVIII}} & \textbf{\textsubscript{M.C.}} &
\textbf{\textsubscript{2017-18}} & \textbf{\textsubscript{1500}} \\
\textbf{\textsubscript{4.}} & \textbf{\textsubscript{Retghat to
Lalghati}} & \textbf{\textsubscript{R.L.}} &
\textbf{\textsubscript{2017-18}} & \textbf{\textsubscript{800}} \\
\textbf{\textsubscript{5.}} & \textbf{\textsubscript{Baghsevania green
belt}} & \textbf{\textsubscript{B.G.}} &
\textbf{\textsubscript{2020-2021}} & \textbf{\textsubscript{300}} \\
\textbf{\textsubscript{6.}} & \textbf{\textsubscript{Shyamapalli Jamburi
maidan}} & \textbf{\textsubscript{S.J.}} &
\textbf{\textsubscript{2021-2022}} & \textbf{\textsubscript{2500}} \\
\textbf{\textsubscript{7.}} & \textbf{\textsubscript{Aura mall Gul mohar
verge}} & \textbf{\textsubscript{A.M.}} &
\textbf{\textsubscript{2018-19}} & \textbf{\textsubscript{280}} \\
\textbf{\textsubscript{8.}} & \textbf{\textsubscript{Chaar Imli}} &
\textbf{\textsubscript{C.I.}} & \textbf{\textsubscript{2017-18}} &
\textbf{\textsubscript{800}} \\
\textbf{\textsubscript{9.}} & \textbf{\textsubscript{near Ashima mall
verge}} & \textbf{\textsubscript{A.S.}} &
\textbf{\textsubscript{2018-19}} & \textbf{\textsubscript{280}} \\
\textbf{\textsubscript{10.}} & \textbf{\textsubscript{Eintkhedi}} &
\textbf{\textsubscript{I.K.}} & \textbf{\textsubscript{2019-2020}} &
\textbf{\textsubscript{1000}} \\
\bottomrule()
\end{longtable}

\textbf{2.2 Selection of Plants:} From each group 4 different plants
were selected and coded according to the species identified.From each
plant, sampling is done as leaves were cut with the help of scissors
from the top, middle, and bottom branchlet of the plant (Ilker Etikan
\emph{et el.,} 2017).

\includegraphics[width=6.49861in,height=3.41667in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image2.png}

\includegraphics[width=6.5in,height=3.59167in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image4.png}\textbf{Fig.2
Photomicrographs of plants with botanical names and codes at 13MP.}

\textbf{2.3.Isolation of Epiphytic Fungi:} Leaf samples from each plant
from the green belt area were collected and coded according to their
plant species identified and brought to laboratory and kept in sterile
conditions. Leaves were cut into discs with the help of cork-borer (5mm
diameter) and placed aseptically at three places in triplicates on
Potato Dextrose Agar (PDA) media. The plates were then incubated at 25±℃
for 5-7 days. After incubation fungal colonies were picked, observed,
and recorded. The pure colonies were sub-cultured on Potato Dextrose
Agar (PDA) media and incubated at 25±℃. (Dubey and Maheshwari.,2020).

\textbf{2.4.Morphological characterization:} For the identification of
fungus, type of conidia, hyphae, conidiophore, and their arrangement
lactophenol cotton Blue mounting was used. The slides were observed
under microscope (Labtech) at 40X magnification. (Dubey and
Maheshwari.,2020).

\textbf{2.5.Serial Purification Cycles:} The sub-culturing process was
repeated sequentially (typically 2 to 3 times) until a uniform colony
morphology with consistent surface texturing, margin shapes, and reverse
pigmentation was achieved across the entire plate.(Bisen, and Verma
1994)

\textbf{2.6.Maintenance of Pure Lines:} Once purity was visually and
microscopically confirmed, the axenic isolates were transferred to agar
slants, incubated until mature sporulation occurred, and then stored at
(4◦ C) to serve as master working stocks for subsequent
characterization.(Cappuccino and Sherman.,2014).

\textbf{2.7.Bio-diversity Analysis of Epiphytes:} Total epiphytic fungal
morphotypes during three different seasons and ten sampling sites were
identified and the diversity, species richness, and evenness of each
morphotype was calculated. The most common diversity indices for
measuring species diversity of a region are the Shannon-Weiner Index and
Ginni- Simpson Index. The Alpha, Beta and Gamma Diversity of morphotypes
was estimated seasonally and geographically by using SPSS Software
4.0,BPMSG Bio-diversity Calculator and MS- Excel Version 2023.

\textbf{2.8.Determination of species diversity by using Shannon--Wiener
diversity index:} It is a popular metric used in ecology. It is based on
Claude Shannon\textquotesingle s formula for entropy and estimates
species diversity. The index considers the number of species living in a
habitat (richness) and their relative abundance (evenness). Shannon
diversity index Formula:-

\emph{\textbf{H=−∑{[}(𝑝i)×log(𝑝i){]}H=−∑{[}(pi)×log(pi){]}}}

where: 𝐻H - Shannon diversity index;

𝑝\textsubscript{i}p\textsubscript{i} - proportion of individuals of i-th
species in a whole community;

∑∑ - sum symbol and

(log - usually the natural logarithm, but the base of the logarithm is
arbitrary(10 and 2 based logarithms were also used).

pi=𝑛𝑁pi=Nn

𝑛n - individuals of a given type/species; and

𝑁N - total number of individuals in a community,

(Shannon et al.,1963)

\textbf{2.9.Determination of species Diversity by Ginni-Simpson Index:}
Community dominated by one or two species is less diverse than one in
which several distinct species have a similar abundance. Simpson's
Diversity Index is a measure of diversity which considers the number of
species present, as well as the relative abundance of each species. As
species richness and evenness increase, so diversity increases.

\begin{longtable}[]{@{}
  >{\raggedright\arraybackslash}p{(\columnwidth - 0\tabcolsep) * \real{1.0000}}@{}}
\toprule()
\begin{minipage}[b]{\linewidth}\raggedright
\includegraphics[width=2.08333in,height=0.775in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image6.gif}
\end{minipage} \\
\midrule()
\endhead
n = the total number of organisms of a particular species \\
N = the total number of organisms of all species \\
\bottomrule()
\end{longtable}

~ (Simpson et al., 1949)

\textbf{2.10.Determination of species Diversity by Berger-Parker Index:}

The Berger-Parker index is~a simple biodiversity index that measures the
proportion of the most abundant species in each sample.~It is calculated
by dividing the number of individuals of the most abundant species (N
max) by the total number of individuals in the sample (N).~The index
ranges from 0 to 1, with 0 indicating complete evenness (no dominance)
and 1 indicating complete dominance (one species makes up the entire
sample. Formula:-

\emph{Berger-Parker index = N\textsubscript{max} / N~}

\textbf{(}Berger \emph{et al.,} 1970)

\textbf{2.11.Determination of Alpha, Beta and Gamma Diversity:}

Alpha diversity refers to diversity on a local scale, describing the
species-diversity (richness) within a functional community. For plants,
alpha diversity is often equated to the count of species identified
during the inventory of a vegetation plot of defined size
(\href{https://pmc.ncbi.nlm.nih.gov/articles/PMC9062518/\#ref49}{Revermann
et al., 2016}).

Beta diversity, on the other hand, describes the amount of
differentiation between species communities. Beta diversity was defined
as the ratio between gamma and alpha diversity
(𝛽~=𝛾/𝛼,~\emph{sensu}~\href{https://pmc.ncbi.nlm.nih.gov/articles/PMC9062518/\#ref65}{Whittaker,
1972}). which captures spatial turnover as well as differences in
diversity between sites.

Gamma diversity describes the overall species diversity across
communities within a larger geographic area. It is defined as diversity
across biogeographic units, such as ecoregions.
(\href{https://pmc.ncbi.nlm.nih.gov/articles/PMC9062518/\#ref11}{Brummitt
et al., 2021}).

\textbf{RESULTS AND DISCUSSION:}

\textbf{3.1.Isolation of Epiphytic Fungi:} 184 viable fungal isolates on
PDA media were obtained from 10 sites during three seasons and
morphologically identified by Lactophenol Cotton blue mounting of fungal
species identified based on type of hyphae, conidiophore and their
arrangement were \emph{Rhizopus, Aspergillus niger, Mucor, Aspergilus
fumigatus, Actinomycetes, Penicillium,Cladosporium,Trichoderma} spp.
\emph{,Fusarium, Curvularia} and \emph{Acremonium} were visualized under
LabTech Microscope at 40X Magnification. The isolated fungal mycobiome
presented distinct cultural and microscopic architectures, pointing to
specialized adaptions for life on the leaf surface. The community was
structurally dominated by filamentous molds belonging to the Ascomycota
and Mucoromycota phyla. Taxonomic evaluation revealed distinct
microscopic traits across the major groups:

\textbf{Table 2: Morphological characteristics of Identified Fungal
isolates obtained from green belt Areas.(Monograph used -Marasinghe, D.
S., et al. 2023., Kidd S.E.et al .2019)}

\begin{longtable}[]{@{}
  >{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.0677}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.1400}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.1400}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.1169}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.1400}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.1400}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.0907}}
  >{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.1646}}@{}}
\toprule()
\begin{minipage}[b]{\linewidth}\raggedright
S.No.
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
Fungal Taxon
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
Colony morphology
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
Reverse Color
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
Texture \& Topography
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
Growth Rate
\end{minipage} & \begin{minipage}[b]{\linewidth}\raggedright
Microscopic Observation
\end{minipage} \\
\midrule()
\endhead
1.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Penicillium sp.}}

\textbf{MiA, AfB,DrA,ScB}} & Bluish-green, grey-green, or yellow-green;
often with a white peripheral border & Pale, yellowish, or deeply
reddish-brown & Velvety, powdery, or cottony; frequently displays radial
furrowing & Rapid to Moderate & oval conidia forming long, unbranched
chains. Septate, hyaline. \textbf{Broom-like} structure
(\emph{penicillus}) with branched metulae and flask-shaped phialides. \\
2

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Fusarium oxysporum}}

\textbf{BmA, EgB1,BhA.}

\textbf{ZmA2,TgC,BmC1,TiB,FgB2}} & White to peach, turning lavender,
pink, or deep purple with age & Pale, violet, dark magenta, or purple &
Cottony, woolly, or fluffy with abundant aerial mycelium & Rapid &
Macroconidia: Sickle-shaped, multi-septate with a foot cell.
Microconidia: Oval, non-septate, in false heads. Septate, hyaline.
Short, simple, or branched conidiophores. \\
3.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Rhizopus sp.}}

\textbf{SaB,EgB1,LsA}

\textbf{TgB}} & White initially, turning grey to yellowish-brown with
dark speckles (sporangia) & White, pale, or greyish & Extremely fluffy,
loose, and "woolly" (resembles cotton candy); fills the petri dish &
Extremely Rapid & Large, round, dark sporangium at apex containing
thousands of round sporangiospores. Aseptate (coenocytic), Long
sporangiophores arise directly from nodes opposite dark, root-like
\textbf{rhizoids}. \\
4.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Aspergillus niger}}

\textbf{MiC, ApC,HcC,TaC1,PeA,PeC,FbA,FbB}} & Carbon black to dark
brown; grainy or powdery & White, pale yellow, or cream & Granular,
dense, and powdery & Rapid & Biseriate phialides covering the entire
vesicle; conidia are globose, rough. Septate, hyaline. Smooth,
thick-walled conidiophore, Jet-black, dense spherical vesicle head \\
5.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Aspergillus terreus}}

\textbf{DlA1,BmC2,FbC}} & Cinnamon-brown, tan, or sandy-orange &
Yellowish to deep brown & Velvety, dense, and finely granular & Rapid &
Biseriate phialides packed compactly, pointing upward parallel to the
stalk (columnar); small, smooth conidia. \\
6.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Aspergillus flavus}}

\textbf{MiB,BhC,AfC,CsC,}} & Yellow-green, olive-green, or bright lime
green & Gold, yellow, or pale maroon & Granular to velvety; may form
dark sclerotia & Rapid & Uniseriate or biseriate phialides covering the
entire vesicle surface; conidia are globose and rough. Septate, hyaline.
hemispherical/dome-shaped vesicle. \\
7.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Aspergillus fumigatus}}

\textbf{EgB2,AfB}} & Smoky grey green to dark blue green & White, tan,
or yellowish & Velvety, powdery, or felt-like & Rapid & Uniseriate
phialides covering only the upper half to two-thirds of the vesicle;
conidia are green and spiky.Septate hyaline \\
8

Isolate

Code. &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Aspergillus flavipes}}

\textbf{MpB}} & Pale yellow, buff, to light beige/tan & Yellowish orange
to purple-brown & Velvety to cottony & Rapid to Moderate & Biseriate
phialides covering a sub-globose vesicle; conidia are small and smooth.
Septate, hyaline. Short, smooth conidiophore with a
flask-shaped/club-shaped vesicle. \\
9.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Aspergillus species}}

\textbf{TaD,McB1,McB3,HiA,FrA,ScC}} & Highly variable; greens, browns,
blacks, yellows, or whites & White, yellow, or brown & Granular,
velvety, or cottony due to dense conidial heads & Rapid & Chains of
conidia produced from phialides arranged radially or columnarily around
the vesicle. Septate, hyaline. \emph{Penicillium}-like structures. \\
10

Isolate

Code. &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\textbf{Aspergillus versicolor}

\textbf{AiA,FgC1}} & Highly variable; pale green, tan, pinkish, yellow,
or multi-colored & Yellow, orange, or deep red & Velvety, dense, and
powdery & Slow to Moderate & Biseriate phialides covering most of the
vesicle; small, rough, globose conidia. Erect conidiophores arising from
a distinct \textbf{foot cell}, terminating in a swollen vesicle. \\
11

Isolate

Code. &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Curvularia sp.}}

\textbf{McB1,McB2,BhB,TcC}} & Olive-green, dark grey, or brownish-black
& Dark brown to black & Woolly, velvety, or fluffy & Rapid & Large,
curved macroconidia (3--5 cells) where the center cell is larger and
darker ("boomerang" shape). \\
12.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Trichoderma resii}}

\textbf{MlC1,FgB1,MpA,TdA2,BhB2,ElB}} & White initially, rapidly
developing dark green to emerald-green tufts & Colorless, pale, or
yellowish & Cottony to granular, often forming concentric rings of green
conidia & Extremely Rapid & Flask-shaped phialides holding clusters of
small, green, globose conidia in sticky heads. \\
13.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Acremonium sp.}}

\textbf{BmB2.FgB,}} & White, grey, pale pink, coral, or salmon &
Colorless, pale pink, or yellow & Moist, glabrous (smooth/waxy)
initially, becoming fine, velvety, or powdery & Slow to Moderate &
Small, oblong conidia bound together in sticky, gelatinous balls at the
tip of the phialides. \\
14.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Cladosporium sp.}}

\textbf{TiA1.AeB1,BvC1}} & Olive-green, velvety brown, to brownish-black
& Dark greenish-black & Velvety, powdery, folded, or heaped & Moderate &
Distinctive "shield cells" (lemon-shaped conidia with dark attachment
scars) forming fragile, branched chains. Dark, branching tree-like
conidiophores \\
15.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Mucor sp.}}

\textbf{TiA2,NlC,BvB1,TaC2,BmB}} & White to light grey, turning dark
grey or yellowish with age & White or pale tan & Fluffy, cottony, and
tall; fills the container (resembles Rhizopus but lacks dark speckles
early on) & Extremely Rapid & Large, round sporangium; a clear columella
(central structure) remains visible after the sporangial wall dissolves.
Randomly branched stalks; lacks rhizoids \\
16.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Gliocladium sp.}}

\textbf{MlC1,MlC2,DpC1,LsB,TgC,PrA}} & White initially, turning dark
green to lawn-green, covering the whole plate & Pale to yellowish & Wet,
slimy, or broadly spreading velvety mat (looks like Trichoderma but more
damp/slimy) & Rapid & Conidia are held together in a large, wet, slimy
green droplet enclosing the whole structure. Convergent phialides
(resembles a wet Penicillium) \\
17.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Colletotrichum sp.}}

\textbf{DpC2,TbA}} & White, grey, to dark grey; often develops orange,
pink, or salmon-colored slimy masses & Pale, orange, or dark grey/black
& Cottony to velvety; characterized by moist conidial masses (acervuli)
& Moderate to Rapid & Oblong, cylindrical, or crescent-shaped conidia
with blunt ends; produced in flat fruiting layers.

Dark, needle-like sterile hairs (setae). \\
18.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Trichophyton sp.}}

\textbf{BmB1,TbB,MhC}} & White, cream, powder-pink, or yellowish &
Highly variable: deep red, yellow, or brown & Powdery, velvety, or
waxy/furrowed & Slow to Moderate & Tear-drop or club-shaped microconidia
arranged along the sides of hyphae

Macroconidia rare. Thin-walled hyphae; spiral coils often present \\
19

Isolate

Code. &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Chaetomium sp.}}

\textbf{ZmA,PeC,FbA,ApB}} & White initially, turning olive-green, grey,
or brown due to mature perithecia & Pale to olive-brown & Cottony,
becoming granular or crusty as fruiting bodies form & Moderate &
Perithecia are covered in characteristic curly, wavy, or branched hairs;
releases lemon-shaped ascospores. \\
20.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Verticillium sp.}}

\textbf{AeB2,ZmA2}} & White to cream initially, turning greyish or black
due to resting structures & Pale or dark blackish-brown & Moist,
velvety, or thin and appressed & Moderate & Oval, single-celled conidia
produced individually at the sharp tips of the whorled phialides. \\
21.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Phoma sp.}}

\textbf{MhB,QgA, ZmB}} & White, grey, olive-green, to dark brown &
Brown, olive-black, or sometimes reddish & Smooth, velvety, or leathery;
develops pepper-like dark spots (pycnidia) embedded in agar & Moderate
to Rapid & Pycnidia have an opening (ostiole) through which thousands of
tiny, oval, single-celled conidia flow out in a mass. \\
22.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Microsporum sp.}}

\textbf{ElB, TcC ,DrB}} & White, buff, tan, to bright yellow orange &
Deep yellow, orange, to brown & Coarse, velvety, or powdery with radial
grooves & Slow to Moderate & Large, spindle-shaped, thick-walled
macroconidia with rough, spiny (echinulate) surfaces and multiple
internal cells. \\
23.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Ulocladium sp.}}

\textbf{AeC BhC,TgA,TbA}} & Dark brown, olive-black, to iron-grey & Dark
brown to black & Velvety, woolly, or dome-shaped fluffy colonies & Rapid
& Obovoid, dark brown dictyoconidia (spores with both transverse and
vertical internal cross-walls); rough-walled. Dark, zig-zag
conidiophores \\
24.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Monascus sp.}}

\textbf{BvA1}} & Surface: Initially white/grey, rapidly turning pink,
yellowish orange, to deep blood-red. & Deep purplish red to dark
brownish red. & Thin, spreading, velvety to slightly cottony; often
restricted with radial furrowing. & Rapid to Moderate. & Produces both
asexual basipetal chains of conidia and sexual, smooth oval ascospores
enclosed within the cleistothecia. Stalked, spherical closed sacs
(cleistothecia). \\
25.

Isolate

Code &
\multicolumn{2}{>{\raggedright\arraybackslash}p{(\columnwidth - 14\tabcolsep) * \real{0.2800} + 2\tabcolsep}}{%
\emph{\textbf{Unidentified}}

\textbf{DlA1 ,ElC2,AiB2}} & Off-White Circular colonies & Cream & Shiny
texture & Rapid & Septa Not clear May be Yeast Rhodotorula or
Actinomycetes or Bacteria Serratia or Pseudomonas \\
\bottomrule()
\end{longtable}

\textbf{3.2.Purification and Morphological Characterization:} The
isolates were evaluated for Surface Topography and Texture such as
velvety, granular, cottony, woolly, or leathery.Colony pigmentation of
the mature spore mass on the surface (e.g., carbon-black, lime-green,
cinnamon-brown, or lavender-pink).Reverse Pigmentation on the underside
of the petri dish to note any exudation of diffusible pigments into the
agar matrix (e.g., bright yellow, deep orange, or colorless).The radial
growth rate was also measured to differentiate between rapid colonizers
(\emph{Mucor} sp. covering a plate in 48 hours) and slow-growing
specialists (\emph{Phoma} sp. or \emph{Acremonium} sp.).

Microscopic slides were prepared using the standard Lactophenol Cotton
Blue (LPCB) teasing or slide-culture method. The lactophenol acts as a
clearing agent and kills the structures, glycerol preserves the
preparation, and cotton blue selectively stains the chitin in the fungal
cell walls, enhancing structural contrast.

\textbf{3.3.Hyphal Morphology:} Differentiated between coenocytic
(non-septate, broad hyphae characteristic of \emph{Mucor sp.}) and
septate hyphae (characteristic of \emph{Aspergillus}, \emph{Fusarium},
and \emph{Penicillium}).

\textbf{3.4.Conidiophore / Sporangiophore Structure:} Examined whether
reproductive stalks were branched or unbranched, smooth or rough-walled,
and evaluated the precise shape of vesicles (globose, dome-shaped, or
clavate).

\textbf{3.5.Spore-Bearing Arrangements:} For example, for
\emph{Aspergillus}, observed the arrangement of sterigmata/phialides
(uniseriate vs. biseriate) and whether they covered the entire vesicle
or were restricted to the upper portion.For \emph{Penicillium}, noted
the signature brush-like conformation (\emph{penicillus}) consisting of
metulae and phialides. For \emph{Trichoderma}, analyzed the clusters of
sticky conidia held on flask-shaped, irregularly branched phialides.

\textbf{3.6.Spore Morphology:} Spore shapes (spherical, lemon-shaped
with attachments scars, or sickle-shaped), texturing (smooth,
echinulate/rough), and septation (single-celled conidia vs.
multi-septate macroconidia in \emph{Fusarium sp.}).

By matching these observed phenotypic profiles against standard
mycological monographs (e.g., Sarah Kidd's \emph{Description of Medical
Fungi}, Barnett, Marasinghe, D. S., et al. 2023 and
Hunter\textquotesingle s \emph{Illustrated Genera of Imperfect Fungi},
and Bisen \& Verma\textquotesingle s manuals), the 184 foliar isolates
were successfully resolved into their respective taxonomic ranks.

\includegraphics[width=6.49974in,height=3.43333in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image7.png}

\includegraphics[width=6.5in,height=3.63125in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image9.png}

\textbf{Fig.3 Photomicrographs of Fungus isolates at 40x magnification
at 13MP and their colony morphology of pure cultures on plates .
(Monograph used for identification-} \textbf{Raper et al., 1965; Samson
2014; Barnett and Hunter, 1987; Domsch and Gams, 2007; Kidd S.E.
2019;Tiwari et al.,2011)}

\includegraphics[width=6.49911in,height=3.75in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image11.png}

\includegraphics[width=6.5in,height=4.53125in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image13.png}\textbf{Fig
4. Photomicrographs of Fungus isolates at 40x magnification at 13MP and
their colony morphology of pure cultures on plates .}

\textbf{3.6.Bio-diversity Analysis of Fungal Morphotypes:}

\textbf{3.6.1.Geographical variation among fungal morphotypes} The
Alpha- Diversity (Seasonal Variance)of fungal morphotypes was
calculated. The Shannon-Index of Fungal morphotypes during winter is
2.16 and rainy season is 2.22 which depicts a higher level of species
diversity, and they are evenly distributed. During summer season Shannon
Index of fungal morphotypes is 1.99 which depicts moderate diversity
with some species with moderate dominance. The Simpson Index during
winter season is 0.12 and rainy season is 0.11 and during summer season
is 0.166 which indicates relatively high diversity and Abundance of
species is relatively even.No single species dominates the community.The
Beta Diversity (Regional) was calculated and the Whittaker Index is 1
which depicts no species differentiation and the Sorensen's similarity
Index is 1 which depicts complete similarity in all regions.The
Geographical diversity was calculated 10 sampling sites and the B.G.
\&M.C. has higher Pi values during winter and Summer season and R.L. has
higher Pi Values in Rainy Season.

\textbf{Fig.5. Geographical Variation of fungal morphotypes during
summer season (a)Rainy (b)Summer (c) Winter}

\textbf{3.6.2.Seasonal Variation among fungal Morphotypes:} The overall
biodiversity is highly dependent on seasonal microclimates.For
high-yield genera like \emph{Mucor} and \emph{Fusarium}, the individual
seasonal bars demonstrate that their massive total numbers are heavily
skewed by optimal monsoon conditions.Warm temperatures coupled with high
relative humidity (RH \textgreater{} 80\%) trigger explosive vegetative
hyphal growth and rapid rain-splash spore dispersal.

On the contrary during winter and summer season lower-yield genera fungi
show localized clusters. \emph{Trichoderma} sp. and \emph{Aspergillus
niger} exhibit strong peaks during cooler intervals, utilizing
persistent morning dewfall to germinate and colonize the leaf boundary
layers. In contrast, the severe thermal bottleneck of the summer season
completely suppresses delicate groups, leaving only highly
thermotolerant species like \emph{Aspergillus terreus} to show active
isolation peaks.

The pie chart shows the frequency \% of fungal morphotypes obtained from
10 sites during three seasons.The highest Abundance frequency \% belongs
to Rainy season followed by winter. The study recorded 65 Aspergillus
isolates from various host plants, with A. niger (28 isolates) being
most prevalent. Clear seasonal variation was observed: winter showed
maximum isolates (49.2\%), followed by rainy (30.7\%) and summer
(20.0\%). A. niger occurred in all seasons, while A. terreus was mainly
a summer species and A. flavus was absent during rainy season. The broad
host range of A. niger across Mango, Neem, Ashoka indicates its
ecological adaptability. \emph{Mucor sp.} was the most frequently
isolated genus, accounting for 28.0\% of total isolates. The dominance
of Mucor is attributable to its rapid growth rate, broad substrate
range, and preference for high moisture conditions prevalent during the
sampling period. This contrasts with studies from drier regions where
Aspergillus and Penicillium typically predominate, suggesting local
climatic factors significantly shape community structure. Fusarium sp.
was the second most abundant genus (19.4\%). Given that several Fusarium
species are soil-borne pathogens causing vascular wilt and root rot,
their high incidence indicates potential Phyto pathological risk to the
surveyed host plants.The genus \emph{Aspergillus c}ollectively comprised
27.6\% of isolates, with A. niger (15.1\%) showing the highest frequency
and broadest host range across 15 plant species including Mangifera
indica, Azadirachta indica, and Saraca asoca. The ecological versatility
of \emph{A. niger} is well known and linked to its production of diverse
extracellular enzymes. A. terreus exhibited distinct seasonal
preference, with 83.3\% of isolates recovered during summer, consistent
with its thermotolerant nature. In contrast, A. flavus was absent in the
rainy season, possibly due to competitive exclusion by fast-growing
Zygomycetes under high humidity.

\includegraphics[width=6.49718in,height=5.75in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image15.png}

\textbf{Fig.6. Heat-map depicting the seasonal variance among fungal
morphotypes.}

\includegraphics[width=6.30756in,height=4.25in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image16.png}

\textbf{Fig.7. Pie- Chart of proportional distribution of Fungal
morphotypes.}

\includegraphics[width=6.49566in,height=2.3125in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image17.png}

\textbf{Fig.8. Lorenz Graph of seasonal variation of Fungal Morphotypes}

\textbf{3.6.3.Fungal Diversity and Host Association Patterns:} The
presence/absence matrix revealed distinct host-fungal association
patterns among isolates from 25 plant species. \emph{Aspergillus niger}
exhibited the widest host range, occurring on 14 different plants
including Mango (Aam), Neem, Jamun, and Peepal, confirming its
ecological versatility as a generalist saprophyte. \emph{Mucor sp.} was
the second most widespread, recorded from 9 hosts, likely due to its
rapid colonization ability under humid conditions. In contrast, \emph{A.
terreus} and \emph{A. fumigatus} showed narrow host specificity,
restricted to Bargad/Peepal and Amla/Imli respectively, suggesting
adaptation to specific host microenvironments. Jamun and Aam supported
the highest fungal diversity with 8 and 7 associated taxa, indicating
these fruit-bearing hosts provide favorable substrates for multiple
fungi. The co-occurrence of potential pathogen \emph{Fusarium sp.} and
biocontrol agent \emph{Trichoderma sp.} on Jamun, Karanj, and Mahua
suggests natural antagonistic interactions. The presence of
opportunistic pathogens \emph{A. flavus} and \emph{A. fumigatus} from
Amla and Aam. Overall, the matrix indicates that while generalist fungi
dominate, significant host-specific interactions exist.

.\includegraphics[width=6.93264in,height=5.13333in]{vertopal_2799d0952d1e48c08f59b39f43c3b423/media/image18.png}
\textbf{Fig.9. Distribution of fungal Morphotypes Across different
plants}

\textbf{3.6.4.Beta Diversity of fungal morphotypes:} Fig. 10 depicts the
alpha and beta diversity indices of fungal morphotypes isolated from
host plants. The Shannon diversity index (H\textquotesingle{} = 3.0)
indicated high species richness and evenness in the fungal community.
Conversely, the low Simpson index (D = 0.15) confirmed high diversity
with no single species dominance. Beta diversity analysis revealed
Whittaker\textquotesingle s index and Sørensen\textquotesingle s
similarity index values near 1.0, suggesting substantial species
turnover between sites and moderate similarity in fungal composition
across regions. These indices collectively indicate a diverse and
heterogeneous mycoflora with distinct regional variation.

\textbf{(a)}

\textbf{(b)}

\textbf{Fig.10.} \textbf{Alpha and beta diversity indices of fungal
morphotypes associated with host plants in Bhopal region. (a)
Shannon-Weaver diversity index (H\textquotesingle) and
Simpson\textquotesingle s dominance index (b)
Whittaker\textquotesingle s beta diversity (βw) and
Sørensen\textquotesingle s similarity index.}

\textbf{CONCLUSION:} The phyllosphere within Bhopal green belts is a
highly organized habitat where community structure is strictly managed
by a three-way interaction: regional seasons control, local surface
chemistry, and host-plant leaf traits govern individual colonization.
These distinct fungal responses can serve as valuable bioindicators for
monitoring urban forest health.

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\end{document}
