Project: Phylloplane Fungi Colonizing Synedrella nodiflora PDF

TRIPURA UNIVERSITY Submitted for the partial fulfillment of M.Sc. 3^rd^ Semester under Department of Botany, Tripura University **Submitted** **by** **Name: Laimi Debbarma** **Roll no.: 230633015** **Registration no. :- 2023-028309** **Microbiology Laboratory** **Department of Botany** ![](...

TRIPURA UNIVERSITY Submitted for the partial fulfillment of M.Sc. 3^rd^ Semester under Department of Botany, Tripura University **Submitted** **by** **Name: Laimi Debbarma** **Roll no.: 230633015** **Registration no. :- 2023-028309** **Microbiology Laboratory** **Department of Botany**  The Project entitled **"An investigation of phylloplane fungi colonizing *Synedrella nodiflora* leaves: a comparative study of young and mature foliage**" has been carried out by **Ms. Laimi Debbarma, bearing roll no.230633015 & registration no. 2023-028309,** as a part of her M.Sc. 3^rd^ Semester in Mycology and Plant pathology under my guidance. Date: (Dr Panna Das) Place: Tripura University, Tripura Supervisor **ACKNOWLEDGEMENTS** I express my deeply felt of gratitude to Dr. Panna Das, Department of Botany, Tripura University for his constant guidance, valuable suggestions during the tenure of my present work. In this respect, I wish to give my sincere gratitude to the Head of Department, Dr. Surochita Basu Department of Botany, Tripura University for her valuable suggestions, kind help and great inspiration. I wish to extend my sincere gratitude to Dr. Badal Kumar Dutta, & Dr. Balasubramaniyam Natesan for providing their encouragements. I also convey my deep gratitude to all the research Scholars of the Department and I am also grateful to all my friends who have always extended their helping hands during the preparation of this project work. I am thankful to all the non- teaching stuff of our department for their immense help. I am grateful to my parents and my family for their support and encouragement. Date: Place: Tripura University, Tripura Laimi Debbarma **\ CONTENTS** SL No. Topic Page No. -------- ----------------------- ---------- 1 Introduction 1 2 Literature Survey 2 - 4 3 Objective 5 4 Materials and methods 6 - 8 5 Result 9-15 6 Discussion 16-17 Work in progress 18 6 References 19-21 **\ ** **INTRODUCTION** Phylloplane refers only to the two surfaces of a plant leaf which constitute a highly complex terrestrial habitat; thus, the study of phylloplane fungi is also concerned with the fungal flora growing on leaf surfaces. The phylloplane is the leaf surface, a unique, dynamic habitat in which a great diversity of microorganisms, including bacteria, yeasts, and fungi are harbored. Phylloplane fungi are especially important members of this microhabitat. They have very important ecological functions and are significantly associated with plant health and productivity (Schmit & Lodge. 2004). *Synedrella nodiflora*, popularly called Cinderella weed, provides a compelling model to study diversity and ecological roles of phylloplane fungi during various developmental stages of the plants. *S. nodiflora* is an herbaceous annual of the Asteraceae family, characterized by its broad, oppositely arranged leaves and abundance of flower heads. Native to Central and South America, Asia this plant has adapted well to disturbed sites, grassland fields, and roadside areas in many tropical and subtropical regions worldwide Phylloplane *S. nodiflora* provides a habitat to a multitude of diverse fungal species that can establish different ecological roles as saprophytes, mutualists, and antagonists. (Holm et al. 1991). Phylloplane fungi can be classified, in terms of their ecological roles, into saprophytic fungi, which play a part in nutrient cycling by decomposing organic refuse like leaf litter and exudates, which enhance soil fertility (Andrews et al. 2000), mutualistic fungi-which interact with plants for a symbiotic relationship-for better nutrient uptake, to stimulate growth, and to increase plant resistance to disease and the environment (Bacon et al. 2000) there is also a good number of phylloplane fungi that are biocontrol agents, as well that compete with, or directly kill, pathogenic microbes thereby playing an important part in economy management of health of plants (Fokkema and Nyckle. 1973). Understanding the diversity and functions of phylloplane fungi on *S. nodiflora* at different growth stages young and mature leaves can yield valuable insights into their functional roles and potential applications in sustainable agriculture and environmental management. The diversity and activity of these fungi are influenced by various factors, including leaf surface characteristics, nutrient availability, and environmental conditions. **LITERATURE SURVEY** **International survey** Estimation and Diversity of Phylloplane fungi on Selected Plants in a Mediterranean--Type Ecosystem in Portugal was studied by J. Ina´cio. The researchers collected leaves from both deciduous trees (*Acer monspessulanum* and *Quercus faginea*) and evergreen shrubs (*Cistus albidus*, *Pistacia lentiscus*, and *Osyris quadripartita*) over two years (Inácio et al. 2002). De Jager studied and investigated the phylloplane microbial community on mango trees, focusing on how various factors such as leaf age, canopy position, seasonality, and chemical spraying affect the density and diversity of microorganisms. Filamentous fungi and yeasts were more abundant in winter and spring Microbial community density and diversity increased with leaf age. Common fungal genera were *Cladosporium* and *Alternaria* (De Jager et al. 2001). The investigation focused on the Phylloplane bacteria on Pisum leaves during the growing season and the early stages of decay. There was minimal filamentous fungal growth on green leaves. However, saprophyte activity sharply increased once the leaves began to senesce, especially on newly dead leaves. During the initial stages, there was a common occurrence of genera such as *Cladosporium, Stemphylium*, and *Alternaria; Cladosporium* and *Stemphylium*s porulated shortly after the leaf died. Changes in leaf physiology were the main reason for the increase in saprophyte activity, but other climatic impacts were also believed to be significant. (Dickinson, 1967). Seasonal changes in the phylloplane of genus *Gnetum* (Gnetaceae) representatives in greenhouse conditions of Pautov et al. (2019) discuss about micromycetes on *Gnetum gnemon* and *Gnetum montanum* leaf surfaces or phylloplane, identifying 17 species that form biofilms and colonize cork warts, causing cell destruction. Population changes with the season will differ in structural organization and biodiversity between the upper and lower leaf surfaces. (Pautovet al. 2019). **National survey** A study was performed to investigate the fungal community in the \"phyllosphere\" and \"phylloplane\" regions of *Oryza sativa\'s* green and recently removed yellow leaves. The phyllosphere-regions of green and yellow leaves have largely different dominating fungal species. The dominant species\' appearance in the \"phyllosphere\" at various phases of plant growth showed the following pattern. During the seedling, pre-flowering, and pre-harvest stages, the in green leaves were *Mucor sp*. *Fussarium sp*, hyaline sterile form, and C*ollectotricum* sp. Pre-harvest *Fussarium oxysporum*in black sterile form and *Penicillium sp*. *Rhizophus stolonifer* hyaline sterile from *Curvularia lunata, C. geniculata* were found in yellow leaves (mishra et al. 1971). Nayak et al. (2017) studied on biodiversity on phylloplane and endophyte fungi from different age group of leaves of medicinal mangrove plant species *Avicennia marina*. Altogether, 25 fungal species of 13 genera were isolated from the mangrove plant, *Avicennia marina*. Among the isolated fungi, *Aspergillus niger, Cochilobolus victoria, Colletotrichum sp., Curvularia lunata, Drechslera sp., Glomerella sp. Fusarium oxysporum, Penicillium chrysogenum, P. oxalicum, Phoma sp., Sordaria sp.* White sterile mycelia were found common in both phylloplane and endophytes. Moist chamber method was found suitable to record more number of fungi than agar plate method. The host relative preference and tissue specification evidence was found between the phylloplane and endophytes based on the fungal community distribution and composition (Nayak et al. 2017) **Northeast India survey** Tanti et al. (2016) study on the phylloplane microflora of various tea cultivars in Assam, focusing on fungi, bacteria, and actinomycetes. Results showed bacterial populations were more dense than fungal populations, with Actinomycetes being sparse. A total of 10 fungal genera were isolated, with *Aspergillus* and *Penicillium* being the dominant mycoflora. Seasonal changes in microbial population were significant. The study found that host characteristics, leaf architecture, chemical environment, and climatic conditions significantly influence the composition and density of phylloplane microflora. Chandra et al. (2022) studied on Ecological diversity of Phylloplane Mycoflora of medicinal plants in Naharlagun, Papumpare District, Arunachal Pradesh, India. The study surveyed the presence of phylloplane fungi on the leaves of eight medicinal plants, *Catharanthus roseus*, *Houttuynia cordata*, *Solanum lycopersicum*, *Murrayakoenigii*, *Eryngium foetidum*, *Clerodendrum glandulosum*, *Ocimum sanctum*, and *Capsicum chinense.* The most abundant identified phylloplane fungi were *Alternaria* sp., *Penicillium sp*., and *Aspergillus sp*., with high relative abundance. Calculation made on relative abundance, species richness, evenness, Shannon-Wiener diversity index, Simpson dominance index, and community similarity index were included in the analysis. The greatest values in the indices mentioned above appeared in *Solanum lycopersicum*, with the second in *Houttuynia cordata, Ocimum sanctum*, and then *Eryngium foetidum*. Comparing different medicinal plants a similar composition of fungal community was indicated using the community similarity index. Ray et al. (2019) studied the fungal diversity affecting the Muga silkworm, *Persea bombycina* (Som), in Goalpara district, Assam, over two years. The quality of Som leaves significantly impacts silk production. The study identifies mycoflora from soil, leaf surfaces, and air with seasonal variations. *Rhizopus stolonifer* dominates rhizosphere, air, and phylloplane, while *Aspergillus niger* dominates non-rhizospheric soil. Joshi et al. (2010) investigated the microbial communities on the leaf surfaces of broad-leaved alder (*Alnus nepalensis*) and needle-leaved khasi pine (*Pinus kesiya*) in the Eastern Himalayas, focusing on how atmospheric dry deposition of roadside pollution influenced these communities dry deposition of roadside pollution in Eastern Himalayas. **OBJECTIVE** The objective of studying phylloplane fungi on young and mature stages of *Synedrella nodiflora* is to understand how these fungi interact with the plant at different growth stages. This includes examining the diversity, abundance of fungi in two different growth stages. By comparing young and mature leaves, researchers can identify any changes in fungal communities and their potential roles. Therefore, the objectives of the project work are as follows: - **To isolate and identify the phylloplane fungi** from the young leaves of *Synedrella nodiflora*. - **To isolate and identify the phylloplane fungi** from the mature leaves of *Synedrella nodiflora*. **MATERIALS AND METHODS** **Collection of samples** To analyse the phylloplane fungi of young and mature stage of *Synederella nodiflora, the leaves samples* of young and mature stage were collected from Tripura university campus \[**91.26075º´/ 23.764363º´´\]**. Leaves samples were carried in closed sterile polythene bags and processed within 24 hours of collection **Fig.1:** Young and mature stage of *Synedrella nodiflora* **PREPARATION OF MEDIA** **Malt Extract Agar (MEA)** Malt Extract media (MEA) are prepared by adding 15 g of malt, 5 g of peptone, and 15 g of agar into 1 liter (1000 ml) distilled water in a conical flask. The media is then sterilized by autoclave at 15 lbs pressure for 30 minutes. Streptomycin is added to the media and the prepared media are poured into previously sterilized petriplates and allowed to set for a few minutes until solid. **Isolation and Morphological Identification of Phylloplane Fungi** Leaf pieces were placed on Petri plates with abaxial surface positioned upward and adaxial surface downward with five adaxial and five abaxial for 1 hour, and then removed with by the method proposed by Santamaria- Bayman (2005). The plates were left at 25°c for the period of 2 to 3 days or longer until the colonies emerged and they were monitored daily for fungal colony growth. The fungi that had emerged from the samples were collected with the help of an inoculation loop and inoculated onto fresh malt extract agar tubes. The transferring of spores to new malt extract agar plates was repeated many times in order to isolate the pure colonies. The fungi were identified with the help of standard manuals \(a) Dematiaceous hypomycetes -- by M.B Ellis; b\) Compendium of soil fungi; by K.H Domsch, W. Gams, Traute Heidi, Anderson; c\) Pictorial Atlas of soil and seed fungi; Morphologies of cultured fungi and key to species - by Tsuneo Watanabe), through cultural characteristics by the help of a compound microscope OLYMPUS CX21i-direct morphology with care on hyphae and reproductive structures. **DATA ANAYLSIS** **Relative frequency (RF)** used to represent fungal density was calculated as \ [\$\$RF\\% = \\frac{\\text{Number\\ of\\ isolates\\ of\\ a\\ species}}{\\text{Total\\ number\\ of\\ isolates}} \\times 100\$\$]{.math.display}\ **The absolute frequency (f)** was calculated as the total number of fungal isolates **RESULT** Relative frequency (RF%) and Absolute frequency(f) Phylloplane fungi isolated from young and matured leaves of *Synedrella nodiflora* collected from Tripura University campus were mentioned in Table no.1&2. A total of 48 fungal isolates belonging to 5 genera of Phylloplane fungi obtained from young leaves of *Synedrella nodiflora* collected from Tripura university campus. In case of young leaves of *Synedrella nodiflora* the highest absolute frequency(f) & relative frequency (RF%) of phylloplane fungi was recorded for *Aspergillus*sp.1 and least for *Penicillium* sp. and *Rhizopus* sp. (Table 1)*.* A total of 70 fungal isolates belonging to 6 genera of Phylloplane fungi obtained from mature leaves of *Synedrella nodiflora* collected from Tripura university campus. In case of mature leaves of *Synedrella nodiflora* the highest absolute frequency (f) & relative frequency (RF%) of phylloplane fungi was recorded for *Penicillium* sp. and least for *Aspergillus* sp.1 and unidentified sp. (Table 2)*.* **Table 1.** Number of colonies of Phylloplane fungi isolated from Abaxial (AB) and Adaxial (AD) surface of the young leaves of *Synedrella nodiflora* with Absolute frequency (f) & Relative frequency (RF) **Fungi** **Colony color** **No. of colonies** **(f)** **RF%** -------------------- ------------------ --------------------- --------- --------- ------- AB AD *Aspergillus* sp.1 Dark brown 13 9 22 45.83 *Aspergillus* sp.2 Yellow 1 2 3 6.25 *Penicillium* sp. Green 2 0 2 4.17 *Rhizopus* sp. White 1 1 2 4.17 White sterile sp. White 10 9 19 39.58  **Fig.2:** Fungal colonies isolated from young leaves of *Synederella nodiflora* **Fig.3:** Microscopic images of pylloplane fungi isolated from the young leaves of *Synederella nodiflora (a) Aspergillus sp.1 (b) Aspergillus sp.2 (c) Rhizopus sp. (d) White sterile sp.(e) Penecillium sp.* **Table 2.** Number of colonies of Phylloplane fungi isolated from Abaxial (AB) and Adaxial (AD) surface of the mature leaves of *Synedrella nodiflora* with Absolute frequency (f) & Relative frequency (RF) **Fungi** **Colony color** **No. of colonies** **(f)** **RF%** --------------------- ------------------ --------------------- --------- --------- ------- AB AD *Aspergillus* sp. Dark brown 0 1 1 1.43 *Cladosporium* sp.1 Grey 5 6 11 15.71 *Fusarium* sp. White 1 1 2 2.86 *Penicillium* sp. Green 21 16 37 52.86 White sterile sp. White 7 11 18 25.71 Unidentified sp. White 1 0 1 1.43  **Fig.4:** Fungal colonies isolated from mature leaves of *Synederella nodiflora* **Fig.5:** Microscopic images of pylloplane fungi isolated from the mature leaves of *Synederella nodiflora (a) Aspergillus sp. (b) White sterile sp. (c) Fusarium sp. (d) Unidentifieed sp. (e) Penicillium sp. (f) cladosporium sp.*  **Fig.6:** Graphical representation of number of colonies of phylloplane fungi isolated from the young and mature leaves of *Synederella nodiflora* **DISCUSSION** The leaf surface habitat that harbours the wide range of both non-pathogenic and pathogenic microbes is called as the phylloplane (Brader et al. **2017**; Kayarkar and Dongarwar, **2019**). Leaves become populated by an assortment of microorganisms from the moment they are formed and continue to sustain microbial populations through their lifetime. In the early stages of life, the leaf is possessed by several restricted or host specific parasites along with primary saprophytes. These fungi obtain their nourishment either from the leaf itself or from the atmosphere. The fungi raid the readily decomposable sugars released from the surface of the leaf, faecal matter and honey dew from the leaf fauna---dead or decomposing parts of the leaf and healthy leaf tissue. The phylloplane fungi start disintegrating the cell walls and swiftly colonize the senescent leaf (Ritpitakphong et al. **2016**; Voříšková and Baldrian, **2013**). A total of 48 fungal isolates belonging to 5 genera of Phylloplane fungi obtained from young leaves of *Synedrella nodiflora* which are two species of *Aspergillus*, *Penicillium* sp., *Rhizopus* sp. and white sterile sp.1. A total of 70 fungal isolates belonging to 6 genera of Phylloplane fungi like *Aspergillus*, *Penicillium* sp., *Fusarium* sp., *Cladosporium* sp., unidentified sp., and white sterile sp.1. obtained from mature leaves of *Synedrella nodiflora* collected from Tripura university campus. Earlier investigations showed Leaf imprint from plants results into the isolations of some common and dominant phylloplane fungi; *Alternaria alternata, Aspergillus niger*, *Aureobasidium pullulans, Cladosporium cladosporioides, Curvularia lunata ,Mucor sp*, *Penicillium sp*, and *Rhizopus sp* (Last and Deighton. 1965; Ruscoe, 1971; Santamaría and Bayman. 2005; Yusifova et al. 2017). However, *Aspergillus niger* has been reported as one of the most dominant inhabitants of aerial leaf surface mycoflora followed by *Cladosporium cladosporioides, Curvulariasp, Alternaria alternata*, *Aureobasidium pullulans*, and *Penicillium* species. Studies have shown that the adaxial surface of leaves has more fungal isolates than the abaxial surface (Vimala and Suriachandraselvan, 2006; De Jager et al. 2001; Ogwu and Osawaru, 2014; Jagiya.2022). Very few fungi can be obtained from young leaves of the plants that are at seedling stage but as plants grow and leaves start to turn senescent the fungal population also exhibits exponential growth (Gulati et al. 2022). Filamentous phylloplane fungi populations increase with leaf age. Low nutrient content on young leaf surfaces may limit early microbial colonization. As leaves mature, nutrient leaching and improved environmental conditions support increased microbial growth (Kirschne., 2015; Mercier and Lindow.2000). Several authors (Last and Deighton. 1965; Ruscoe, 1971; Santamaría and Bayman. 2005; Yusifova et al. 2017) have reported *Aspergillus*, *Mucor*, *Penicillium*, and *Rhizopus* as phylloplane fungi which is similar to present study. Plant phylloplane represents a unique ecological niche, actively colonized by various filamentous and yeast fungi of different taxonomic groups. Among the fungi that can be identified on the surface of plants, there are saprotrophic species that are capable of developing in such difficult environmental conditions---high insolation, low humidity, and lack of nutrients. We propose to use the term "epiphytic fungi" specifically for this unique ecological group, the importance of which is now generally recognized. These epiphytic fungi can play an important role in the life of the host plant, influencing its metabolism and exhibiting antagonistic actions toward parasitic fungi. In addition, epiphytic fungi are likely to be an important initial step in the subsequent decomposition of leaf litter, participating in the cycle of material cycling. From a practical point of view, fungi living in the phylloplane and exposed to a complex of stress factors can be a potential source of important biological substances, thereby having significant potential for biotechnology (Tsarelunga and Blagoveschenskaya, 2024). **WORK IN PROGRESS** To isolate and identify phylloplane fungi during the winter season from *Synederella nodiflora.* **REFERENCE** Andrews, J. H., & Harris, R. F. (2000). The ecology and biogeography of microorganisms on plant surfaces. Annual Review of Phytopathology, 38, 145-180.** ** Bacon, C. W., & White Jr, J. F. (2000). Physiological adaptations in the evolution of endophytism in the Clavicipitaceae. In Microbial endophytes (pp. 251-276). CRC Press. Brader, G.; Compant, S.; Vescio, K.; Mitter, B.; Trognitz, F.; Ma, L.J.; Sessitsch, A. Ecology and genomic insights into plant-pathogenic and plant-nonpathogenic endophytes. Annu. Rev. Phytopathol. 2017, 55, 61--83. Chanda, D., Devi, S. P., & Das, N. 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