First Record of Phycopeltis Arundinacea (Trentepohliaceae, Chlorophyta) on Oil Palm (Elaeis Guineensis) in Malaysia: Morphological Characterisation and Evidence of Lichen-Associated Structures

First Record of Phycopeltis Arundinacea (Trentepohliaceae, Chlorophyta) on Oil Palm (Elaeis Guineensis) in Malaysia: Morphological Characterisation and Evidence of Lichen-Associated Structures | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First Record of Phycopeltis Arundinacea (Trentepohliaceae, Chlorophyta) on Oil Palm (Elaeis Guineensis) in Malaysia: Morphological Characterisation and Evidence of Lichen-Associated Structures Raimathy Kanavedee, Nurul Athirah Zaifornoor, Nur Hafizah Norhata, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9540797/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Epiphytic red algae are commonly observed on oil palm leaves in humid tropical plantations; however, their taxonomic identity and ecological significance in Malaysia remain insufficiently documented. In other crops, similar algae are often associated with lichens, composite organisms formed through a symbiotic relationship between a photobiont and a mycobiont. Foliicolous lichens specifically develop on leaf surfaces of host plants. This study aimed to identify and characterise the epiphytic alga colonising oil palm ( Elaeis guineensis ) fronds, quantify symptom severity across frond ages and estates, and evaluate canopy-level spatial patterns. Leaf samples were collected from six oil palm estates in Peninsular Malaysia. Symptom severity was assessed using the Leaf Doctor digital image analysis application. Drone-acquired red, green, and blue imagery was analysed to compute the Visible Difference Vegetation Index. Morphological characterisation was conducted using stereomicroscopy, light microscopy, and scanning electron microscopy. Isolation trials were performed on Bold’s Basal Medium with and without Indole-3-Acetic Acid supplementation. Symptom severity increased significantly with frond age, with Fronds 25 and 33 exhibiting the highest colonisation levels (up to 84%). Vegetation index analysis revealed spatial clustering of affected areas. Microscopic observations confirmed discoid epiphytic thalli with radiating filaments and sporangial structures consistent with Phycopeltis arundinacea as the dominant photobiont. Trebouxia-like cells and fungal associations were also observed, suggesting possible lichen-like interactions. The leaf epidermis remained intact, distinguishing this species from pathogenic Cephaleuros spp. This study provides the first confirmed report of Phycopeltis arundinacea on oil palm in Malaysia and presents an integrated approach for epiphytic algal characterisation. Epiphytic Algae Oil Palm Phycopeltis arundinacea Morphological Identification Lichenisation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Within the family Trentepohliaceae (Chlorophyta), the genera Phycopeltis and Cephaleuros are frequently encountered in humid tropical environments. Although both taxa may produce reddish to brownish epiphytic colonies on leaf surfaces, they differ fundamentally in ecological behaviour and pathogenic potential (Brooks et al., 2015 ; Huang et al., 2023 ). Phycopeltis species are strictly epiphytic, forming disc-shaped thalli composed of uniseriate, radiating filaments that adhere to the leaf cuticle without penetrating host tissues. In contrast, Cephaleuros species are parasitic algae capable of producing haustorial structures that invade epidermal cells, often resulting in necrotic leaf spots (Neustupa, 2005 ; Brooks et al., 2015 ). Morphologically, Phycopeltis colonies may resemble Cephaleuros in field conditions, leading to potential misidentification. Without microscopic confirmation, epiphytic colonies can be mistakenly diagnosed as pathogenic infections, potentially resulting in unnecessary management interventions (Widariyanto, Astari & Sugandi, 2024 ). Therefore, accurate morphological characterisation is essential for correct taxonomic identification and ecological interpretation. Oil palm ( Elaeis guineensis Jacq.) is one of the most economically important plantation crops in tropical regions, contributing substantially to global vegetable oil production and Malaysia’s agricultural economy (Corley & Tinker, 2016 ; FAO, 2023; MPOB, 2023; USDA, 2023). The productivity of oil palm depends on the physiological integrity of its fronds, which serve as the primary photosynthetic organs supporting fresh fruit bunch development (Taiz et al., 2015 ). Consequently, biological factors affecting leaf surfaces may influence canopy health and yield performance. Epiphytic microorganisms are important components of tropical phyllosphere ecosystems (Lindow & Brandl, 2003 ; Vorholt, 2012 ). Members of the family Trentepohliaceae, including Phycopeltis , are widely distributed in humid environments and commonly colonise plant leaf surfaces (Liu et al., 2022 ). These algae typically form discoid, epiphyllous thalli that remain confined to the leaf surface without tissue penetration (Thompson & Wujek, 1997 ). Although orange-to-reddish epiphytic growth is frequently observed in Malaysian oil palm plantations, systematic studies that integrate morphological identification, quantitative severity assessment, and spatial evaluation remain limited. In addition, algal–fungal interactions in tropical systems are ecologically significant, particularly in lichen symbioses where the fungal partner (mycobiont) and photosynthetic partner (photobiont) form a mutualistic association (Zuo et al., 2023 ; Schulz et al., 2022 ). However, the occurrence and structural characteristics of such associations in oil palm plantation environments have not been thoroughly investigated. To the best of our knowledge, this is the first study in Malaysia to provide confirmed morphological identification of Phycopeltis arundinacea associated with oil palm plantations, integrating quantitative severity assessment and microscopic validation. This work establishes a baseline record for distinguishing epiphytic Phycopeltis from pathogenic Cephaleuros species in plantation systems. Therefore, this study aimed to (i) confirm the taxonomic identity of the epiphytic alga colonising oil palm fronds through detailed morphological characterisation, (ii) quantify symptom severity across frond developmental stages and estates using digital assessment tools, and (iii) evaluate spatial patterns of colonisation to better understand its distribution within plantation canopies. MATERIALS AND METHODS Leaf Sampling Leaf samples were collected from six oil palm estates located in northern and southern Peninsular Malaysia. Estates from both northern and southern regions were included to ensure geographic representation. All sampling was conducted using a standardised protocol to maintain consistency across locations. From each selected palm (n = 3), leaflets were sampled from four frond positions (F9, F17, F25, and F33), with six leaflets collected per frond, resulting in 18 leaflet samples per frond position (n = 18). Symptom Documentation and Sample Processing Leaflets were collected from four frond positions (Fronds 9, 17, 25, and 33) representing different developmental stages. Samples were obtained from three palms per estate. For each frond, twelve leaflets were randomly selected. Six leaflets per frond were used for digital severity assessment, while the remaining six were subjected to laboratory analysis. These samples were rinsed with tap water, surface-sterilised using 70% ethanol, and subsequently washed with distilled water. Midribs were removed, and leaf tissues were cut into approximately 5 cm segments. Between six and ten segments were placed in labelled sterile bags for isolation and microscopic examination. Drone Imagery and Visible Difference Vegetation Index (VDVI) Analysis Unmanned aerial vehicle (UAV) imagery was acquired at one representative estate to assess canopy-level variation associated with epiphytic colonisation. High-resolution red, green, and blue (RGB) images were processed to generate orthomosaic maps, from which reflectance values were extracted to calculate the Visible Difference Vegetation Index (VDVI) as follows: \(\:VDVI=\frac{\left(2G−R−B\right)}{\left(2G+R+B\right)}\) where R, G, and B represent the reflectance values of the red, green, and blue channels, respectively. VDVI maps were generated and classified into severity categories based on pixel intensity thresholds, ranging from no infection to severe infection. The index was further used to evaluate spatial variation and identify clustering patterns of colonised areas within the canopy. Leaf Doctor Severity Assessment Leaflets measuring 8 cm × 4 cm were photographed under uniform lighting conditions using a light box. Images were analysed using the Leaf Doctor mobile application. Healthy tissue colours were selected to establish baseline references, after which the software classified pixels as healthy or symptomatic. The threshold was adjusted manually to refine classification, and the percentage of symptomatic tissue was calculated for each sample with measurements conducted in six replicates (n = 45). ITS Amplicon Sequencing To confirm the taxonomic identity of the epiphytic algae observed during the field survey, supplemental symptomatic leaf samples were collected from Batu Lintang Estate for molecular characterisation. Leaf tissues were homogenised in liquid nitrogen using a TissueLyser II system, and genomic DNA was extracted from three biological replicates using a KingFisher™ Flex automated platform with NucleoMag Plant kit (MACHEREY-NAGEL, Germany) and NucleoMag® NGS Clean-up (MACHEREY-NAGEL, Germany). DNA extracts were pooled, purified, and quantified prior to amplification. The ITS2 region of ribosomal DNA was amplified using ITS3/ITS4 primers and sequenced on an Illumina MiSeq platform (paired - end). Bioinformatics analysis was performed using the EzBioCloud MTP pipeline (CJ Bioscience, South Korea). Raw sequencing reads were subjected to quality control, including filtering of low-quality reads and removal of chimeric sequences. After quality filtering, an average of 22,003 reads per sample was retained for downstream analysis. High-quality reads were clustered into operational taxonomic units (OTUs). Taxonomy assignment was subsequently performed against UNITE database (version 9.0) using VSEARCH based on 97% similarity to determine the dominant algal taxa present. In Vitro Culture Bold’s Basal Medium (BBM) agar was prepared by dissolving BBM premix in distilled water, adjusting the pH to 6.8, and solidifying with agar before sterilisation. For experimental treatments, the medium was supplemented with Indole-3-Acetic Acid (IAA) at 2mL L − 1 , prepared from a 0.1% (w/v) stock solution. Approximately 5 mm leaf segments from symptomatic tissues were aseptically transferred onto culture media. Plates were incubated under continuous light at 28°C for up to two months, and algal growth was monitored periodically. Morphological Examination Leaf samples were initially examined using a stereomicroscope to observe surface colonisation patterns. For detailed cellular observations, symptomatic tissues were further processed for light microscopy, and algal material was gently removed for closer examination. For scanning electron microscopy (SEM), samples were prepared using the Critical Point Drying (CPD) method (Talbot & White, 2013 ). Samples were first cut into 1 mm 3 pieces and fixed in 2% (v/v) glutaraldehyde for 12–24 h at 4°C. Following fixation, samples were rinsed with 0.1 M phosphate buffered saline (PBS) for 10 min, repeated three times to ensure removal of residual fixative. Dehydration was carried out through a graded ethanol series (30%, 50%, 70%, 80%, 90% and 100%), with each step lasting 10 min. The 100% ethanol step was repeated three times to ensure complete dehydration. Samples were then transferred to a critical point dryer and processed accordingly to standard CPD protocols. Dried specimens were mounted onto aluminium stubs using double-sided conductive tape and sputter-coated with a thin layer of gold (approximately 5–10 nm). SEM imaging was performed using a Thermo Scientific Quattro S Field Emission Scanning Electron Microscope (FE-SEM; Thermo Fisher Scientific, USA). The instrument was operated under high vacuum conditions at an accelerating voltage of 5–15 kV. Images were captured using secondary electron (SE) and backscattered electron (BSE) detectors to observe surface morphology and structural features. Statistical Analysis Differences in symptom severity among frond age classes and estates were analysed using the Kruskal–Wallis test, followed by Dunn's post-hoc pairwise comparisons with Bonferroni adjustment. All statistical analyses were performed using the dunn.test package in R (version 4.5.1). A total of 432 samples were included in the analysis, with 18 samples collected for each symptom severity category (healthy, mild, moderate, and severe) per estate, across six estates, yielding 72 samples per estate and 108 samples per symptom severity category. Statistical significance was set at p < 0.05. RESULTS Remote Sensing and Severity Assessment Drone-based VDVI analysis revealed spatial heterogeneity in canopy condition. Areas with lower VDVI values corresponded with visually observed epiphytic colonisation, and affected zones exhibited clustered distribution patterns, suggesting potential influence of local microclimatic conditions (Fig. 1 ). Leaf Doctor analysis (Table 1) demonstrated a consistent increase in symptom severity with frond maturity across all estates. Older fronds (F25 and F33) exhibited substantially higher proportions of reddish-orange symptomatic tissue compared to younger fronds (F9 and F17). Severity levels differed among estates, with Telok Sengat and BMR recording the highest values, while Bekoh and Paloh showed comparatively lower levels. Buntar and Landak displayed intermediate severity. Symptomatic patches were generally distributed along the lamina but were more concentrated in the central region of mature fronds (F25 and F33). Quantitative assessment (Table 2 ) confirmed this age-related trend. At F9, symptomatic tissue remained low across estates (1–4%). Severity increased moderately at F17 (13–22%), with the highest values recorded in Buntar and Landak. A pronounced increase was observed at F25 (32–63%), with BMR recording the highest percentage (63%). The greatest severity occurred at F33, ranging from 50–84% across estates. Non-parametric statistical analysis (Kruskal–Wallis test followed by Dunn’s post hoc test with Bonferroni adjustment) revealed significant differences among estates within each severity category. Under mild conditions, Bekoh showed significantly lower severity compared to Buntar and Landak (adjusted p < 0.001). In the moderate category, Paloh differed significantly from BMR and several other estates (adjusted p < 0.001). Under severe conditions, Paloh exhibited significantly higher severity than Bekoh, BMR, and Telok Sengat (adjusted p < 0.001) (Table 3 ). These results indicate clear spatial variation in colonisation intensity across locations. In Vitro Culture of the Photobiont Leaf tissues containing epiphytic colonies were inoculated onto Bold’s Basal Medium (BBM), with and without Indole-3-Acetic Acid (IAA), under continuous 24-hour light conditions to evaluate algal growth response. The primary filamentous alga ( Phycopeltis ) did not establish growth under the in vitro conditions tested. However, unicellular green algal cells consistent with Trebouxia successfully proliferated in culture (Fig. 3 ). On standard BBM medium without IAA supplementation, visible algal growth required approximately 30–40 days. In contrast, cultures grown on BBM supplemented with IAA exhibited detectable growth within 14 days. IAA-treated cultures showed earlier colony expansion and pigmentation changes compared to non-supplemented media, for all samples across different estates. Continuous light exposure supported algal development, whereas extended dark periods were associated with reduced algal proliferation and increased fungal overgrowth. These observations suggest that light regime and auxin supplementation influenced in vitro establishment and growth rate of the unicellular photobiont. Molecular Analysis Preliminary internal transcribed spacer (ITS) amplicon sequencing revealed consistent detection of sequences affiliated with the class Trebouxiophyceae and the order Trentepohliales (Fig. 4 ). Members of Trentepohliales belong to the family Trentepohliaceae , which includes genera such as Cephaleuros and Phycopeltis , both of which are commonly associated with algal colonisation in tropical crops. The molecular results were consistent with the morphological characteristics observed in symptomatic tissues, particularly the presence of discoid, epiphyllous thalli with radiating filaments typical of Phycopeltis . The concordance between sequencing data and microscopic observations supports the taxonomic identification of the organism detected in this study. Stereomicroscopic Examination Stereomicroscopic analysis revealed progressive colonisation patterns across frond ages and estates (Table 4 ). Surface observations enabled detailed visualisation of lesion colour, size, and distribution on the leaf lamina. In all estates, younger fronds (F9 and F17) exhibited early stages of colonisation. At F9, only one to two small reddish-orange spots were observed, while F17 showed scattered, discrete colonies that remained non-coalescent. At F25, increased lesion density was evident in several estates, with patches beginning to cluster and expand. This trend was more pronounced in Estate Bekoh and Paloh, where colonies started to merge. By F33, extensive coalescence was observed in most estates, resulting in large, continuous patches of reddish-orange and green algal growth covering substantial portions of the leaf surface. A similar developmental progression was recorded in Buntar and Landak, although lesion expansion appeared more pronounced at earlier stages compared to Bekoh and Paloh. Overall, lesion size and coverage increased consistently with frond maturity. Light Microscopy Light microscopic examination of samples collected from all six estates (Buntar, Bekoh, Paloh, Landak, Telok Sengat, and BMR) revealed consistent morphological characteristics (Fig. 5 ). Despite differences in severity recorded through stereomicroscopic assessment and Leaf Doctor analysis, the cellular and thallus structures were uniform across locations. The algae formed well-defined, circular discoid thalli firmly attached to the leaf surface. Each thallus consisted of uniseriate filaments radiating from a central region, producing a pseudoparenchymatous disc-like structure. Vegetative cells were rectangular to slightly elongated, measuring approximately 6–9 µm in width and up to 17 µm in length. Cells were radially arranged and frequently exhibited pseudodichotomous branching. Chloroplasts were parietal and fragmented, and pyrenoids were not observed. Pigmentation varied from green to reddish-orange, with darker colouration typically concentrated in the central portion of the colony. Spherical to ovoid sporangia (8–10 µm in diameter and 10–14 µm in length) were observed, often originating from enlarged vegetative cells. Some central cells appeared empty, consistent with zoosporangial release. Importantly, the leaf epidermis remained intact in all examined samples, and no penetration or invasive structures were detected. Based on the combination of discoid thallus formation, radial uniseriate filaments, pseudodichotomous branching, and sporangial morphology, the specimens were identified as Phycopeltis arundinacea (Trentepohliales, Trentepohliaceae). The uniform morphology across estates indicates that variation in symptom severity was not attributable to species-level differences. In Vivo Lichen-Associated Structures Microscopic examination of epidermal peel samples revealed the presence of algal cells, fungal hyphae, and lichen-like structures at varying developmental stages (Fig. 6 ). Two types of green algal cells were observed in association with the structures: discoid filamentous algae consistent with Phycopeltis and unicellular cells resembling Trebouxia . Phycopeltis was frequently observed in a free-living state on the leaf surface. In several samples, multicellular, disc-shaped thalli composed of tightly branched, radially organised filaments were detected. These thalli were covered by a network of superficial fungal hyphae, forming structures resembling early-stage lichenisation. The unicellular green algal cells ( Trebouxia -like) were found scattered in clusters, sometimes in proximity to fungal hyphae. However, the exact origin and nature of the algal–fungal association could not be determined based on morphological observation alone. Microscopic and scanning electron microscopic (SEM) examinations confirmed the colonisation of symptomatic oil palm leaf surfaces by Phycopeltis arundinacea (Fig. 7 ). Colonies appeared as compact, flat, disc-shaped thalli composed of radially arranged, branched uniseriate filaments firmly attached to the epidermal surface. Vegetative cells contained chloroplasts and carotenoid pigments, producing the characteristic green-to-orange appearance observed during field symptom assessment. Terminal and intercalary sporangial structures were frequently detected, developing from vegetative cells as thick-walled reproductive units containing multiple internal spores, indicating active asexual propagation. Several empty central cells were observed, consistent with spore release. SEM analysis further demonstrated strong surface adherence and extensive lateral expansion of the thalli across the cuticular layer, confirming the strictly epiphytic nature of the organism. Gametangial structures were occasionally observed but were less common than sporangial forms, suggesting that natural populations predominantly reproduce asexually. In some samples, superficial fungal hyphae were found in close proximity to algal filaments, forming lichenisation-like arrangements; however, the precise nature of this algal–fungal association could not be determined based solely on morphological evidence. DISCUSSION The present study provides the first systematic documentation of epiphytic red algal colonisation on oil palm leaves in Malaysia and confirms the presence of Phycopeltis arundinacea across multiple estates based on morphological, microscopic, and preliminary molecular evidence. Symptom severity increased with frond maturity, with older fronds exhibiting greater surface coverage than younger fronds, a pattern consistent with previous observations that epiphytic members of Trentepohliales preferentially colonise mature foliage due to prolonged exposure and favourable microclimatic stability (Nelson, 2008 ; Brooks et al., 2015 ). Estate-level variation further suggests that local humidity, canopy structure, and leaf wetness duration play decisive roles in colonisation intensity, as similarly reported for tropical epiphytic algae in plantation systems (Neustupa, 2005 ; Widariyanto et al., 2024 ). Microscopic and SEM analyses revealed discoid, radially arranged thalli composed of uniseriate branched filaments with intercalary and terminal sporangia, corresponding closely with established descriptions of Phycopeltis arundinacea (Neustupa, 2005 ). The absence of epidermal penetration confirms its predominantly epiphytic habit and distinguishes it from pathogenic Trentepohliaceae such as Cephaleuros , which invade host tissues and cause necrosis (Nelson, 2008 ). However, the occasional presence of superficial fungal hyphae closely associated with algal filaments forming lichenisation-like configurations introduces an additional layer of ecological complexity. Members of Phycopeltis have previously been reported as photobionts in foliicolous lichen systems (Reshma & Job, 2019 ), and molecular detection of Trebouxiophyceae sequences in preliminary analyses suggests the possible coexistence of additional green algal partners such as Trebouxia , a well-known lichen phycobiont (Poquita-Du., 2026). Although the present work did not include multilocus phylogenetic confirmation of fungal partners, these observations highlight the necessity to investigate more deeply the extent and nature of lichenisation, including the structural organisation of algal–fungal interfaces, the specificity of symbiotic pairing, and the spatial distribution of potential dual photobionts. Importantly, this study should be regarded as a preliminary investigation that provides only a glimpse into the morphological diversity and reproductive strategies of this foliicolous system. The dominance of sporangial structures suggests active asexual propagation in the field, yet the absence of clearly defined sexual reproductive stages raises questions regarding life cycle regulation under natural versus in vitro conditions. Further research should therefore focus on characterising the developmental stages of the lichen in controlled culture systems, including co-cultivation experiments to determine whether Phycopeltis functions as a primary phycobiont or occurs independently with opportunistic fungal association. Detailed ultrastructural studies, coupled with molecular barcoding of both algal and fungal components, would clarify symbiotic specificity and potential coexistence with Trebouxia . In addition, physiological assays examining nutrient exchange, photosynthetic performance, and stress tolerance under varying humidity and light regimes would help elucidate the reciprocal influence between the algal partner, associated fungi, and the oil palm host. Such integrative approaches are necessary to move beyond surface-level morphological description and to understand whether the observed associations represent transient epiphytism, facultative lichenisation, or a more structured mutualistic system. Collectively, these findings establish a foundational framework for future ecological, taxonomic, and symbiotic investigations into foliicolous algal–fungal interactions in tropical plantation environments. Declarations Author Contribution Raimathy - Project leader and conducted the experiments Athirah- Observed microscopy and media preparation Hafizah- Microscopy and media preparationAdzhar- Drone images Suet Yee-Statistician, analyse data Laura-VDVI imaging Helmay- Sample collection from different areaTan Swee Sian- Reviewing the experiments and approval and reviewing the manuscript Tasren- Approving the research, Director of research, reviewing the manuscript ACKNOWLEDGEMENT The authors thank the principals, Kuala Lumpur Kepong Berhad and Boustead Plantations Berhad, for their financial support. References Brooks, F. Rindi, F. Suto, Y. Ohtani, S. & Green, M. 2015. The Trentepohliales (Ulvophyceae, Chlorophyta): An Unusual Algal Order and Its Novel Plant Pathogen— Cephaleuros . Plant Disease , 99(6): 740–753. Corley, R. H. V., & Tinker, P. B. (2016). The Oil Palm (5th ed.). Wiley-Blackwell. Food and Agriculture Organization (FAO). (2023). FAOSTAT statistical database . FAO. Huang, Y.-C., Tsai, C.-Y., Wang, C.-L. (2023). Host invasion type is a phylogenetically conserved characteristic of Cephaleuros. Plant Disease , 107(10), 3222–3229. Lindow, S. E., & Brandl, M. T. (2003). Microbiology of the phyllosphere. Applied and Environmental Microbiology , 69(4), 1875–1883. Liu, B.-W., Li, S.-Y., Zhu, H., & Liu, G.-X. (2022). Phyllosphere eukaryotic microalgal communities in rainforests: Drivers and diversity. Plant Diversity , 45(1), 45–53. https://doi.org/10.1016/j.pld.2022.08.006 Malaysian Palm Oil Board (MPOB). (2023). Malaysian oil palm statistics 2023 . MPOB, Malaysia. Nelson, S. C. 2008. Cephaleuros Species, The Plant-Parasitic Green Algae. University of Hawai‘i, Plant Disease Publication. Neustupa, J. 2005. Investigations on The Genus Phycopelti s (Trentepohliaceae, Chlorophyta) From Southeast Asia, Including Descriptions of Two New Species. Cryptogamie Algologie, 26(3): 229–238. Poquita-Du RC, Otte J, Herrmann N, Büchel C, Schmitt I. Members of the lichen photobiont genus Trebouxia show species-specific photophysiological and transcriptome-level responses to high light. J Exp Bot. 2026;77(2):609–624. doi: 10.1093/jxb/eraf419 . PMID: 40973683; PMCID: PMC12794234. Reshma, R. S. & Job, J. 2019. An Introductory Study on Foliicolous Lichen Found on Common Trees in Kerala, India: Characterization of Its Symbiotic Partners. International Journal of Current Advanced Research, 8(4): 18462–18468. Schulz, M., Schmitt, I., Weber, D., & Dal Grande, F. (2022). Fungal Host Affects Photosynthesis in a Lichen Holobiont. Journal of Fungi, 8(12), 1267. Taiz, L., Zeiger, E., Møller, I. M., & Murphy, A. (2015). Plant Physiology and Development (6th ed.). Sinauer Associates. Talbot, M. J., & White, R. G. (2013). Methanol fixation of plant tissue for scanning electron microscopy improves preservation of tissue morphology and dimensions. Plant Methods , 9, 36. https://doi.org/10.1186/1746-4811-9-36 . Thompson, R. H., & Wujek, D. E. (1997).Trentepohliales: Cephaleuros, Phycopeltis and related genera. Science Publishers. United States Department of Agriculture (USDA). (2023). Oilseeds: World markets and trade. USDA Foreign Agricultural Service. Vorholt, J. A. (2012). Microbial life in the phyllosphere. Nature Reviews Microbiology, 10, 828–840. Widariyanto, R. Astari, S. & Sugandi, A. 2024. Morphological Characteristics and Identification of Algae Species on Oil Palm Leaves in an Oil Palm Estate in Riau, Indonesia. IOP Conference Series: Earth and Environmental Science, 1308(1): 012030. Zuo, Y.-B., Han, D.-Y., Wang, Y.-Y., Yang, Q.-X., Ren, Q., Liu, X.-Z., & Wei, X.-L. (2023). Fungal–Algal Association Drives Lichens’ Mutualistic Symbiosis: A Case Study with Trebouxia-Related Lichens. Plants, 12(17), 3172 Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9540797","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":630623931,"identity":"8e610b0a-94af-4134-b7b4-0f85c11e23b4","order_by":0,"name":"Raimathy Kanavedee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYFACxgYgcYCBH84mTkvCAQbJBuK1gABQi8EBYrWYtx9u3fDzx53EzccPP/v4cweDPL/YAdbNPHi0yJxJbLvZk/AscduZNOPZvGcYDGfOTmC7jU+LBENi2w2ehMOJ2w4kGDMztjEkGNwmpIX/YdvNP0Atm/uff2b8SZQWicS22yBbNkjkGDPwEqflYdttmbTDxjNuvClm5j0jAfQL0Hdz8Dos/dnNNzaHZfv70zcz/txhI88vnXzsxhs8WmDAsQFqBAModpjwOQwG7FF4jD+I0DIKRsEoGAUjBgAA0otWiyYsBnkAAAAASUVORK5CYII=","orcid":"","institution":"Advanced Agriecological Research Sdn. 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Bhd","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Adzhar","lastName":"Jaafar","suffix":""},{"id":630623937,"identity":"623706b7-a806-48b8-8daa-7317c6f9a939","order_by":4,"name":"Jia Xin Ong","email":"","orcid":"","institution":"Advanced Agriecological Research Sdn. Bhd","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"Xin","lastName":"Ong","suffix":""},{"id":630623939,"identity":"cb848202-4fdb-4412-ac68-13da899117b8","order_by":5,"name":"Suet Yee Tan","email":"","orcid":"","institution":"Advanced Agriecological Research Sdn. Bhd","correspondingAuthor":false,"prefix":"","firstName":"Suet","middleName":"Yee","lastName":"Tan","suffix":""},{"id":630623940,"identity":"e6457b3c-9168-4dd7-9b6e-9b60dc1502ab","order_by":6,"name":"Zhi Chin Lin Laura","email":"","orcid":"","institution":"Advanced Agriecological Research Sdn. Bhd","correspondingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"Chin Lin","lastName":"Laura","suffix":""},{"id":630623943,"identity":"41033516-807c-4b44-a889-25d1e4d88fc3","order_by":7,"name":"Mohd Helmay Husaini Mohd Zin","email":"","orcid":"","institution":"Advanced Agriecological Research Sdn. Bhd","correspondingAuthor":false,"prefix":"","firstName":"Mohd","middleName":"Helmay Husaini Mohd","lastName":"Zin","suffix":""},{"id":630623945,"identity":"c218a8b0-857c-4a63-a819-45074919cd99","order_by":8,"name":"Swee Sian Tan","email":"","orcid":"","institution":"Advanced Agriecological Research Sdn. Bhd","correspondingAuthor":false,"prefix":"","firstName":"Swee","middleName":"Sian","lastName":"Tan","suffix":""},{"id":630623946,"identity":"5a5b1e2c-ced3-4aff-a7c4-a3ea9cb0eea9","order_by":9,"name":"Zi Yen Chen","email":"","orcid":"","institution":"Advanced Agriecological Research Sdn. Bhd","correspondingAuthor":false,"prefix":"","firstName":"Zi","middleName":"Yen","lastName":"Chen","suffix":""},{"id":630623947,"identity":"95f6f0f5-8191-429e-9405-bba8019c6cae","order_by":10,"name":"Tasren Nazir Mahamooth","email":"","orcid":"","institution":"Advanced Agriecological Research Sdn. Bhd","correspondingAuthor":false,"prefix":"","firstName":"Tasren","middleName":"Nazir","lastName":"Mahamooth","suffix":""}],"badges":[],"createdAt":"2026-04-27 11:10:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9540797/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9540797/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108073231,"identity":"e1d09230-50fe-45e3-b734-de98b201d78a","added_by":"auto","created_at":"2026-04-29 06:18:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":192649,"visible":true,"origin":"","legend":"\u003cp\u003eVisible Difference Vegetation Index (VDVI) map of oil palm canopy derived from drone imagery. VDVI values ranged from −1 to +1. Warmer colours (yellow–red) indicate lower vegetation vigour and higher epiphytic algae severity, while cooler colours (green–dark blue) represent healthier canopy areas. Severity classification was categorised based on VDVI thresholds and validated using ground-truth disease severity (%) quantified by Leaf Doctor application.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/3b1d1e4c1e7734de239e74af.png"},{"id":108181779,"identity":"ce0f2916-1114-4537-bf1b-770c7e7d170a","added_by":"auto","created_at":"2026-04-30 08:58:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101786,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage Symptom Severity (%) at different frond ages across different estates.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/9b582720ed1b8998bedede58.png"},{"id":108073235,"identity":"0bbbc448-4fa1-4bc5-8b0f-f392cc481b5b","added_by":"auto","created_at":"2026-04-29 06:18:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1076844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003egrowth of unicellular photobiont\u003cem\u003e (Trebouxia sp.) \u003c/em\u003eon BBM supplemented with Indole-3-Acetic Acid under continuous light. Green and orange pigmentation was observed after 14 days of incubation (20X Magnification).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/e4d4267f08ced43c82dc8b2c.png"},{"id":108181450,"identity":"c12986b8-8541-4f37-b3f3-c946ca677dcd","added_by":"auto","created_at":"2026-04-30 08:58:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135835,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of ITS amplicon sequences showing taxonomic affiliation within \u003cem\u003eTrebouxiophyceae\u003c/em\u003e and \u003cem\u003eTrentepohliales\u003c/em\u003edetected in symptomatic oil palm samples.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/8e1d3532937130ae1895ec15.png"},{"id":108181544,"identity":"2dd5ca13-c200-463b-9739-98f10cfd07c9","added_by":"auto","created_at":"2026-04-30 08:58:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1142053,"visible":true,"origin":"","legend":"\u003cp\u003eLight microscopic observation for Leaf Samples across 6 Estates (Magnification: 40X)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/70a63df8d8119163d0fa8e71.png"},{"id":108490949,"identity":"ae6d08a2-7c4a-4835-9f50-54b992039bce","added_by":"auto","created_at":"2026-05-05 09:50:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1735968,"visible":true,"origin":"","legend":"\u003cp\u003eA: Light Microscopy 40X Magnification. Discoid filamentous green alga (\u003cem\u003ePhycopeltis\u003c/em\u003e) and unicellular green algal cells (\u003cem\u003eTrebouxia\u003c/em\u003e-like) observed on the leaf surface. B: \u003cem\u003ePhycopeltis\u003c/em\u003e in proximity to lichen-associated structures. C: Disc-shaped algal thallus covered by superficial fungal hyphae forming lichen-like architecture.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/d9d7b93e9159056746738a5b.png"},{"id":108073238,"identity":"27f33f46-a076-4ba7-8c93-70768a7dff39","added_by":"auto","created_at":"2026-04-29 06:18:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1227513,"visible":true,"origin":"","legend":"\u003cp\u003eScanning Electron Micrographs (SEM) of \u003cem\u003ePhycopeltis arundinacea\u003c/em\u003ecolonising oil palm leaf surfaces and associated fungal interactions. A: Low-magnification view of extensive epiphytic colonisation demostration lateral expansion of the adult algal thallus across the cuticular surface. Bar = 400 µm. B: Well-developed radiating filamentous thallus with multiple terminal and intercalary sporangial structures distributed across the colony surface, indicating active asexual reproduction. Bar = 200 µm. C: Sporangial structures (arrowed) indicating asexual propagation of \u003cem\u003ePhycopeltis sp\u003c/em\u003e. Bar = 40 µm. D: Gametangium (arrowed) of \u003cem\u003eP. arundinacea. \u003c/em\u003eBar = 10 µm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/f51d048d438d0d732c207f83.png"},{"id":108804370,"identity":"4c820cb0-758a-43e7-a7ef-87557d77301a","added_by":"auto","created_at":"2026-05-08 15:19:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5817804,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/62eb0da3-88f3-462b-9e12-c7fa6a19d9bf.pdf"},{"id":108073232,"identity":"9ffa2560-657c-4539-95b3-76939a4ad8d2","added_by":"auto","created_at":"2026-04-29 06:18:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26701812,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-9540797/v1/d1ad27cff1c510fa92bb1e5c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFirst Record of Phycopeltis Arundinacea (Trentepohliaceae, Chlorophyta) on Oil Palm (Elaeis Guineensis) in Malaysia: Morphological Characterisation and Evidence of Lichen-Associated Structures\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eWithin the family Trentepohliaceae (Chlorophyta), the genera \u003cem\u003ePhycopeltis\u003c/em\u003e and \u003cem\u003eCephaleuros\u003c/em\u003e are frequently encountered in humid tropical environments. Although both taxa may produce reddish to brownish epiphytic colonies on leaf surfaces, they differ fundamentally in ecological behaviour and pathogenic potential (Brooks et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003ePhycopeltis\u003c/em\u003e species are strictly epiphytic, forming disc-shaped thalli composed of uniseriate, radiating filaments that adhere to the leaf cuticle without penetrating host tissues. In contrast, \u003cem\u003eCephaleuros\u003c/em\u003e species are parasitic algae capable of producing haustorial structures that invade epidermal cells, often resulting in necrotic leaf spots (Neustupa, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Brooks et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Morphologically, \u003cem\u003ePhycopeltis\u003c/em\u003e colonies may resemble \u003cem\u003eCephaleuros\u003c/em\u003e in field conditions, leading to potential misidentification. Without microscopic confirmation, epiphytic colonies can be mistakenly diagnosed as pathogenic infections, potentially resulting in unnecessary management interventions (Widariyanto, Astari \u0026amp; Sugandi, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, accurate morphological characterisation is essential for correct taxonomic identification and ecological interpretation.\u003c/p\u003e \u003cp\u003eOil palm (\u003cem\u003eElaeis guineensis\u003c/em\u003e Jacq.) is one of the most economically important plantation crops in tropical regions, contributing substantially to global vegetable oil production and Malaysia\u0026rsquo;s agricultural economy (Corley \u0026amp; Tinker, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; FAO, 2023; MPOB, 2023; USDA, 2023). The productivity of oil palm depends on the physiological integrity of its fronds, which serve as the primary photosynthetic organs supporting fresh fruit bunch development (Taiz et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consequently, biological factors affecting leaf surfaces may influence canopy health and yield performance.\u003c/p\u003e \u003cp\u003eEpiphytic microorganisms are important components of tropical phyllosphere ecosystems (Lindow \u0026amp; Brandl, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Vorholt, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Members of the family Trentepohliaceae, including \u003cem\u003ePhycopeltis\u003c/em\u003e, are widely distributed in humid environments and commonly colonise plant leaf surfaces (Liu et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These algae typically form discoid, epiphyllous thalli that remain confined to the leaf surface without tissue penetration (Thompson \u0026amp; Wujek, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Although orange-to-reddish epiphytic growth is frequently observed in Malaysian oil palm plantations, systematic studies that integrate morphological identification, quantitative severity assessment, and spatial evaluation remain limited.\u003c/p\u003e \u003cp\u003eIn addition, algal\u0026ndash;fungal interactions in tropical systems are ecologically significant, particularly in lichen symbioses where the fungal partner (mycobiont) and photosynthetic partner (photobiont) form a mutualistic association (Zuo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Schulz et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the occurrence and structural characteristics of such associations in oil palm plantation environments have not been thoroughly investigated.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, this is the first study in Malaysia to provide confirmed morphological identification of \u003cem\u003ePhycopeltis arundinacea\u003c/em\u003e associated with oil palm plantations, integrating quantitative severity assessment and microscopic validation. This work establishes a baseline record for distinguishing epiphytic \u003cem\u003ePhycopeltis\u003c/em\u003e from pathogenic \u003cem\u003eCephaleuros\u003c/em\u003e species in plantation systems.\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to (i) confirm the taxonomic identity of the epiphytic alga colonising oil palm fronds through detailed morphological characterisation, (ii) quantify symptom severity across frond developmental stages and estates using digital assessment tools, and (iii) evaluate spatial patterns of colonisation to better understand its distribution within plantation canopies.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLeaf Sampling\u003c/h2\u003e \u003cp\u003eLeaf samples were collected from six oil palm estates located in northern and southern Peninsular Malaysia. Estates from both northern and southern regions were included to ensure geographic representation. All sampling was conducted using a standardised protocol to maintain consistency across locations. From each selected palm (n\u0026thinsp;=\u0026thinsp;3), leaflets were sampled from four frond positions (F9, F17, F25, and F33), with six leaflets collected per frond, resulting in 18 leaflet samples per frond position (n\u0026thinsp;=\u0026thinsp;18).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSymptom Documentation and Sample Processing\u003c/h3\u003e\n\u003cp\u003eLeaflets were collected from four frond positions (Fronds 9, 17, 25, and 33) representing different developmental stages. Samples were obtained from three palms per estate. For each frond, twelve leaflets were randomly selected.\u003c/p\u003e \u003cp\u003eSix leaflets per frond were used for digital severity assessment, while the remaining six were subjected to laboratory analysis. These samples were rinsed with tap water, surface-sterilised using 70% ethanol, and subsequently washed with distilled water. Midribs were removed, and leaf tissues were cut into approximately 5 cm segments. Between six and ten segments were placed in labelled sterile bags for isolation and microscopic examination.\u003c/p\u003e\n\u003ch3\u003eDrone Imagery and Visible Difference Vegetation Index (VDVI) Analysis\u003c/h3\u003e\n\u003cp\u003eUnmanned aerial vehicle (UAV) imagery was acquired at one representative estate to assess canopy-level variation associated with epiphytic colonisation. High-resolution red, green, and blue (RGB) images were processed to generate orthomosaic maps, from which reflectance values were extracted to calculate the Visible Difference Vegetation Index (VDVI) as follows:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:VDVI=\\frac{\\left(2G\u0026minus;R\u0026minus;B\\right)}{\\left(2G+R+B\\right)}\\)\u003c/span\u003e\u003c/span\u003ewhere R, G, and B represent the reflectance values of the red, green, and blue channels, respectively.\u003c/p\u003e \u003cp\u003eVDVI maps were generated and classified into severity categories based on pixel intensity thresholds, ranging from no infection to severe infection. The index was further used to evaluate spatial variation and identify clustering patterns of colonised areas within the canopy.\u003c/p\u003e\n\u003ch3\u003eLeaf Doctor Severity Assessment\u003c/h3\u003e\n\u003cp\u003eLeaflets measuring 8 cm \u0026times; 4 cm were photographed under uniform lighting conditions using a light box. Images were analysed using the Leaf Doctor mobile application. Healthy tissue colours were selected to establish baseline references, after which the software classified pixels as healthy or symptomatic. The threshold was adjusted manually to refine classification, and the percentage of symptomatic tissue was calculated for each sample with measurements conducted in six replicates (n\u0026thinsp;=\u0026thinsp;45).\u003c/p\u003e\n\u003ch3\u003eITS Amplicon Sequencing\u003c/h3\u003e\n\u003cp\u003eTo confirm the taxonomic identity of the epiphytic algae observed during the field survey, supplemental symptomatic leaf samples were collected from Batu Lintang Estate for molecular characterisation. Leaf tissues were homogenised in liquid nitrogen using a TissueLyser II system, and genomic DNA was extracted from three biological replicates using a KingFisher\u0026trade; Flex automated platform with NucleoMag Plant kit (MACHEREY-NAGEL, Germany) and NucleoMag\u0026reg; NGS Clean-up (MACHEREY-NAGEL, Germany).\u003c/p\u003e \u003cp\u003eDNA extracts were pooled, purified, and quantified prior to amplification. The ITS2 region of ribosomal DNA was amplified using ITS3/ITS4 primers and sequenced on an Illumina MiSeq platform (paired - end). Bioinformatics analysis was performed using the EzBioCloud MTP pipeline (CJ Bioscience, South Korea). Raw sequencing reads were subjected to quality control, including filtering of low-quality reads and removal of chimeric sequences. After quality filtering, an average of 22,003 reads per sample was retained for downstream analysis. High-quality reads were clustered into operational taxonomic units (OTUs). Taxonomy assignment was subsequently performed against UNITE database (version 9.0) using VSEARCH based on 97% similarity to determine the dominant algal taxa present.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn Vitro Culture\u003c/h2\u003e \u003cp\u003eBold\u0026rsquo;s Basal Medium (BBM) agar was prepared by dissolving BBM premix in distilled water, adjusting the pH to 6.8, and solidifying with agar before sterilisation. For experimental treatments, the medium was supplemented with Indole-3-Acetic Acid (IAA) at 2mL L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, prepared from a 0.1% (w/v) stock solution.\u003c/p\u003e \u003cp\u003eApproximately 5 mm leaf segments from symptomatic tissues were aseptically transferred onto culture media. Plates were incubated under continuous light at 28\u0026deg;C for up to two months, and algal growth was monitored periodically.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMorphological Examination\u003c/h3\u003e\n\u003cp\u003eLeaf samples were initially examined using a stereomicroscope to observe surface colonisation patterns. For detailed cellular observations, symptomatic tissues were further processed for light microscopy, and algal material was gently removed for closer examination.\u003c/p\u003e \u003cp\u003eFor scanning electron microscopy (SEM), samples were prepared using the Critical Point Drying (CPD) method (Talbot \u0026amp; White, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Samples were first cut into 1 mm\u003csup\u003e3\u003c/sup\u003e pieces and fixed in 2% (v/v) glutaraldehyde for 12\u0026ndash;24 h at 4\u0026deg;C. Following fixation, samples were rinsed with 0.1 M phosphate buffered saline (PBS) for 10 min, repeated three times to ensure removal of residual fixative.\u003c/p\u003e \u003cp\u003eDehydration was carried out through a graded ethanol series (30%, 50%, 70%, 80%, 90% and 100%), with each step lasting 10 min. The 100% ethanol step was repeated three times to ensure complete dehydration. Samples were then transferred to a critical point dryer and processed accordingly to standard CPD protocols. Dried specimens were mounted onto aluminium stubs using double-sided conductive tape and sputter-coated with a thin layer of gold (approximately 5\u0026ndash;10 nm).\u003c/p\u003e \u003cp\u003eSEM imaging was performed using a Thermo Scientific Quattro S Field Emission Scanning Electron Microscope (FE-SEM; Thermo Fisher Scientific, USA). The instrument was operated under high vacuum conditions at an accelerating voltage of 5\u0026ndash;15 kV. Images were captured using secondary electron (SE) and backscattered electron (BSE) detectors to observe surface morphology and structural features.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eDifferences in symptom severity among frond age classes and estates were analysed using the Kruskal\u0026ndash;Wallis test, followed by Dunn's post-hoc pairwise comparisons with Bonferroni adjustment. All statistical analyses were performed using the \u003cem\u003edunn.test\u003c/em\u003e package in R (version 4.5.1). A total of 432 samples were included in the analysis, with 18 samples collected for each symptom severity category (healthy, mild, moderate, and severe) per estate, across six estates, yielding 72 samples per estate and 108 samples per symptom severity category. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eRemote Sensing and Severity Assessment\u003c/h2\u003e\n \u003cp\u003eDrone-based VDVI analysis revealed spatial heterogeneity in canopy condition. Areas with lower VDVI values corresponded with visually observed epiphytic colonisation, and affected zones exhibited clustered distribution patterns, suggesting potential influence of local microclimatic conditions (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eLeaf Doctor analysis (Table\u0026nbsp;1) demonstrated a consistent increase in symptom severity with frond maturity across all estates. Older fronds (F25 and F33) exhibited substantially higher proportions of reddish-orange symptomatic tissue compared to younger fronds (F9 and F17). Severity levels differed among estates, with Telok Sengat and BMR recording the highest values, while Bekoh and Paloh showed comparatively lower levels. Buntar and Landak displayed intermediate severity. Symptomatic patches were generally distributed along the lamina but were more concentrated in the central region of mature fronds (F25 and F33).\u003c/p\u003e\n \u003cp\u003eQuantitative assessment (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) confirmed this age-related trend. At F9, symptomatic tissue remained low across estates (1\u0026ndash;4%). Severity increased moderately at F17 (13\u0026ndash;22%), with the highest values recorded in Buntar and Landak. A pronounced increase was observed at F25 (32\u0026ndash;63%), with BMR recording the highest percentage (63%). The greatest severity occurred at F33, ranging from 50\u0026ndash;84% across estates.\u003c/p\u003e\n \u003cp\u003eNon-parametric statistical analysis (Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s post hoc test with Bonferroni adjustment) revealed significant differences among estates within each severity category. Under mild conditions, Bekoh showed significantly lower severity compared to Buntar and Landak (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the moderate category, Paloh differed significantly from BMR and several other estates (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Under severe conditions, Paloh exhibited significantly higher severity than Bekoh, BMR, and Telok Sengat (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results indicate clear spatial variation in colonisation intensity across locations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eIn Vitro Culture of the Photobiont\u003c/h2\u003e\n \u003cp\u003eLeaf tissues containing epiphytic colonies were inoculated onto Bold\u0026rsquo;s Basal Medium (BBM), with and without Indole-3-Acetic Acid (IAA), under continuous 24-hour light conditions to evaluate algal growth response.\u003c/p\u003e\n \u003cp\u003eThe primary filamentous alga (\u003cem\u003ePhycopeltis\u003c/em\u003e) did not establish growth under the in vitro conditions tested. However, unicellular green algal cells consistent with \u003cem\u003eTrebouxia\u003c/em\u003e successfully proliferated in culture (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eOn standard BBM medium without IAA supplementation, visible algal growth required approximately 30\u0026ndash;40 days. In contrast, cultures grown on BBM supplemented with IAA exhibited detectable growth within 14 days. IAA-treated cultures showed earlier colony expansion and pigmentation changes compared to non-supplemented media, for all samples across different estates.\u003c/p\u003e\n \u003cp\u003eContinuous light exposure supported algal development, whereas extended dark periods were associated with reduced algal proliferation and increased fungal overgrowth. These observations suggest that light regime and auxin supplementation influenced in vitro establishment and growth rate of the unicellular photobiont.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMolecular Analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePreliminary internal transcribed spacer (ITS) amplicon sequencing revealed consistent detection of sequences affiliated with the class \u003cem\u003eTrebouxiophyceae\u003c/em\u003e and the order \u003cem\u003eTrentepohliales\u003c/em\u003e (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Members of \u003cem\u003eTrentepohliales\u003c/em\u003e belong to the family \u003cem\u003eTrentepohliaceae\u003c/em\u003e, which includes genera such as \u003cem\u003eCephaleuros\u003c/em\u003e and \u003cem\u003ePhycopeltis\u003c/em\u003e, both of which are commonly associated with algal colonisation in tropical crops.\u003c/p\u003e\n \u003cp\u003eThe molecular results were consistent with the morphological characteristics observed in symptomatic tissues, particularly the presence of discoid, epiphyllous thalli with radiating filaments typical of \u003cem\u003ePhycopeltis\u003c/em\u003e. The concordance between sequencing data and microscopic observations supports the taxonomic identification of the organism detected in this study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eStereomicroscopic Examination\u003c/h2\u003e\n \u003cp\u003eStereomicroscopic analysis revealed progressive colonisation patterns across frond ages and estates (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Surface observations enabled detailed visualisation of lesion colour, size, and distribution on the leaf lamina.\u003c/p\u003e\n \u003cp\u003eIn all estates, younger fronds (F9 and F17) exhibited early stages of colonisation. At F9, only one to two small reddish-orange spots were observed, while F17 showed scattered, discrete colonies that remained non-coalescent.\u003c/p\u003e\n \u003cp\u003eAt F25, increased lesion density was evident in several estates, with patches beginning to cluster and expand. This trend was more pronounced in Estate Bekoh and Paloh, where colonies started to merge. By F33, extensive coalescence was observed in most estates, resulting in large, continuous patches of reddish-orange and green algal growth covering substantial portions of the leaf surface.\u003c/p\u003e\n \u003cp\u003eA similar developmental progression was recorded in Buntar and Landak, although lesion expansion appeared more pronounced at earlier stages compared to Bekoh and Paloh. Overall, lesion size and coverage increased consistently with frond maturity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eLight Microscopy\u003c/h2\u003e\n \u003cp\u003eLight microscopic examination of samples collected from all six estates (Buntar, Bekoh, Paloh, Landak, Telok Sengat, and BMR) revealed consistent morphological characteristics (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Despite differences in severity recorded through stereomicroscopic assessment and Leaf Doctor analysis, the cellular and thallus structures were uniform across locations.\u003c/p\u003e\n \u003cp\u003eThe algae formed well-defined, circular discoid thalli firmly attached to the leaf surface. Each thallus consisted of uniseriate filaments radiating from a central region, producing a pseudoparenchymatous disc-like structure. Vegetative cells were rectangular to slightly elongated, measuring approximately 6\u0026ndash;9 \u0026micro;m in width and up to 17 \u0026micro;m in length. Cells were radially arranged and frequently exhibited pseudodichotomous branching. Chloroplasts were parietal and fragmented, and pyrenoids were not observed.\u003c/p\u003e\n \u003cp\u003ePigmentation varied from green to reddish-orange, with darker colouration typically concentrated in the central portion of the colony. Spherical to ovoid sporangia (8\u0026ndash;10 \u0026micro;m in diameter and 10\u0026ndash;14 \u0026micro;m in length) were observed, often originating from enlarged vegetative cells. Some central cells appeared empty, consistent with zoosporangial release. Importantly, the leaf epidermis remained intact in all examined samples, and no penetration or invasive structures were detected.\u003c/p\u003e\n \u003cp\u003eBased on the combination of discoid thallus formation, radial uniseriate filaments, pseudodichotomous branching, and sporangial morphology, the specimens were identified as \u003cem\u003ePhycopeltis arundinacea\u003c/em\u003e (Trentepohliales, Trentepohliaceae). The uniform morphology across estates indicates that variation in symptom severity was not attributable to species-level differences.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eIn Vivo Lichen-Associated Structures\u003c/h2\u003e\n \u003cp\u003eMicroscopic examination of epidermal peel samples revealed the presence of algal cells, fungal hyphae, and lichen-like structures at varying developmental stages (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Two types of green algal cells were observed in association with the structures: discoid filamentous algae consistent with \u003cem\u003ePhycopeltis\u003c/em\u003e and unicellular cells resembling \u003cem\u003eTrebouxia\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ePhycopeltis\u003c/em\u003e was frequently observed in a free-living state on the leaf surface. In several samples, multicellular, disc-shaped thalli composed of tightly branched, radially organised filaments were detected. These thalli were covered by a network of superficial fungal hyphae, forming structures resembling early-stage lichenisation.\u003c/p\u003e\n \u003cp\u003eThe unicellular green algal cells (\u003cem\u003eTrebouxia\u003c/em\u003e-like) were found scattered in clusters, sometimes in proximity to fungal hyphae. However, the exact origin and nature of the algal\u0026ndash;fungal association could not be determined based on morphological observation alone.\u003c/p\u003e\n \u003cp\u003eMicroscopic and scanning electron microscopic (SEM) examinations confirmed the colonisation of symptomatic oil palm leaf surfaces by \u003cem\u003ePhycopeltis arundinacea\u003c/em\u003e (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Colonies appeared as compact, flat, disc-shaped thalli composed of radially arranged, branched uniseriate filaments firmly attached to the epidermal surface. Vegetative cells contained chloroplasts and carotenoid pigments, producing the characteristic green-to-orange appearance observed during field symptom assessment. Terminal and intercalary sporangial structures were frequently detected, developing from vegetative cells as thick-walled reproductive units containing multiple internal spores, indicating active asexual propagation. Several empty central cells were observed, consistent with spore release. SEM analysis further demonstrated strong surface adherence and extensive lateral expansion of the thalli across the cuticular layer, confirming the strictly epiphytic nature of the organism. Gametangial structures were occasionally observed but were less common than sporangial forms, suggesting that natural populations predominantly reproduce asexually. In some samples, superficial fungal hyphae were found in close proximity to algal filaments, forming lichenisation-like arrangements; however, the precise nature of this algal\u0026ndash;fungal association could not be determined based solely on morphological evidence.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study provides the first systematic documentation of epiphytic red algal colonisation on oil palm leaves in Malaysia and confirms the presence of \u003cem\u003ePhycopeltis arundinacea\u003c/em\u003e across multiple estates based on morphological, microscopic, and preliminary molecular evidence. Symptom severity increased with frond maturity, with older fronds exhibiting greater surface coverage than younger fronds, a pattern consistent with previous observations that epiphytic members of Trentepohliales preferentially colonise mature foliage due to prolonged exposure and favourable microclimatic stability (Nelson, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Brooks et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Estate-level variation further suggests that local humidity, canopy structure, and leaf wetness duration play decisive roles in colonisation intensity, as similarly reported for tropical epiphytic algae in plantation systems (Neustupa, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Widariyanto et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Microscopic and SEM analyses revealed discoid, radially arranged thalli composed of uniseriate branched filaments with intercalary and terminal sporangia, corresponding closely with established descriptions of \u003cem\u003ePhycopeltis arundinacea\u003c/em\u003e (Neustupa, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The absence of epidermal penetration confirms its predominantly epiphytic habit and distinguishes it from pathogenic Trentepohliaceae such as \u003cem\u003eCephaleuros\u003c/em\u003e, which invade host tissues and cause necrosis (Nelson, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, the occasional presence of superficial fungal hyphae closely associated with algal filaments forming lichenisation-like configurations introduces an additional layer of ecological complexity. Members of \u003cem\u003ePhycopeltis\u003c/em\u003e have previously been reported as photobionts in foliicolous lichen systems (Reshma \u0026amp; Job, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and molecular detection of Trebouxiophyceae sequences in preliminary analyses suggests the possible coexistence of additional green algal partners such as \u003cem\u003eTrebouxia\u003c/em\u003e, a well-known lichen phycobiont (Poquita-Du., 2026). Although the present work did not include multilocus phylogenetic confirmation of fungal partners, these observations highlight the necessity to investigate more deeply the extent and nature of lichenisation, including the structural organisation of algal\u0026ndash;fungal interfaces, the specificity of symbiotic pairing, and the spatial distribution of potential dual photobionts.\u003c/p\u003e \u003cp\u003eImportantly, this study should be regarded as a preliminary investigation that provides only a glimpse into the morphological diversity and reproductive strategies of this foliicolous system. The dominance of sporangial structures suggests active asexual propagation in the field, yet the absence of clearly defined sexual reproductive stages raises questions regarding life cycle regulation under natural versus in vitro conditions. Further research should therefore focus on characterising the developmental stages of the lichen in controlled culture systems, including co-cultivation experiments to determine whether \u003cem\u003ePhycopeltis\u003c/em\u003e functions as a primary phycobiont or occurs independently with opportunistic fungal association. Detailed ultrastructural studies, coupled with molecular barcoding of both algal and fungal components, would clarify symbiotic specificity and potential coexistence with \u003cem\u003eTrebouxia\u003c/em\u003e. In addition, physiological assays examining nutrient exchange, photosynthetic performance, and stress tolerance under varying humidity and light regimes would help elucidate the reciprocal influence between the algal partner, associated fungi, and the oil palm host. Such integrative approaches are necessary to move beyond surface-level morphological description and to understand whether the observed associations represent transient epiphytism, facultative lichenisation, or a more structured mutualistic system. Collectively, these findings establish a foundational framework for future ecological, taxonomic, and symbiotic investigations into foliicolous algal\u0026ndash;fungal interactions in tropical plantation environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRaimathy - Project leader and conducted the experiments Athirah- Observed microscopy and media preparation Hafizah- Microscopy and media preparationAdzhar- Drone images Suet Yee-Statistician, analyse data Laura-VDVI imaging Helmay- Sample collection from different areaTan Swee Sian- Reviewing the experiments and approval and reviewing the manuscript Tasren- Approving the research, Director of research, reviewing the manuscript\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eThe authors thank the principals, Kuala Lumpur Kepong Berhad and Boustead Plantations Berhad, for their financial support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrooks, F. Rindi, F. Suto, Y. Ohtani, S. \u0026amp; Green, M. 2015. The Trentepohliales (Ulvophyceae, Chlorophyta): An Unusual Algal Order and Its Novel Plant Pathogen\u0026mdash;\u003cem\u003eCephaleuros\u003c/em\u003e. \u003cem\u003ePlant Disease\u003c/em\u003e, 99(6): 740\u0026ndash;753.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorley, R. H. V., \u0026amp; Tinker, P. B. (2016). \u003cem\u003eThe Oil Palm\u003c/em\u003e (5th ed.). Wiley-Blackwell.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFood and Agriculture Organization (FAO). (2023). \u003cem\u003eFAOSTAT statistical database\u003c/em\u003e. FAO.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, Y.-C., Tsai, C.-Y., Wang, C.-L. (2023). Host invasion type is a phylogenetically conserved characteristic of Cephaleuros. \u003cem\u003ePlant Disease\u003c/em\u003e, 107(10), 3222\u0026ndash;3229.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindow, S. E., \u0026amp; Brandl, M. T. (2003). Microbiology of the phyllosphere. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e, 69(4), 1875\u0026ndash;1883.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, B.-W., Li, S.-Y., Zhu, H., \u0026amp; Liu, G.-X. (2022). Phyllosphere eukaryotic microalgal communities in rainforests: Drivers and diversity. \u003cem\u003ePlant Diversity\u003c/em\u003e, 45(1), 45\u0026ndash;53. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pld.2022.08.006\u003c/span\u003e\u003cspan address=\"10.1016/j.pld.2022.08.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalaysian Palm Oil Board (MPOB). (2023). \u003cem\u003eMalaysian oil palm statistics 2023\u003c/em\u003e. MPOB, Malaysia.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNelson, S. C. 2008. Cephaleuros Species, The Plant-Parasitic Green Algae. University of Hawai\u0026lsquo;i, Plant Disease Publication.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeustupa, J. 2005. Investigations on The Genus \u003cem\u003ePhycopelti\u003c/em\u003es (Trentepohliaceae, Chlorophyta) From Southeast Asia, Including Descriptions of Two New Species. Cryptogamie Algologie, 26(3): 229\u0026ndash;238.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoquita-Du RC, Otte J, Herrmann N, B\u0026uuml;chel C, Schmitt I. Members of the lichen photobiont genus Trebouxia show species-specific photophysiological and transcriptome-level responses to high light. J Exp Bot. 2026;77(2):609\u0026ndash;624. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jxb/eraf419\u003c/span\u003e\u003cspan address=\"10.1093/jxb/eraf419\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 40973683; PMCID: PMC12794234.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReshma, R. S. \u0026amp; Job, J. 2019. An Introductory Study on Foliicolous Lichen Found on Common Trees in Kerala, India: Characterization of Its Symbiotic Partners. International Journal of Current Advanced Research, 8(4): 18462\u0026ndash;18468.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulz, M., Schmitt, I., Weber, D., \u0026amp; Dal Grande, F. (2022). Fungal Host Affects Photosynthesis in a Lichen Holobiont. Journal of Fungi, 8(12), 1267.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaiz, L., Zeiger, E., M\u0026oslash;ller, I. M., \u0026amp; Murphy, A. (2015). Plant Physiology and Development (6th ed.). Sinauer Associates.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalbot, M. J., \u0026amp; White, R. G. (2013). Methanol fixation of plant tissue for scanning electron microscopy improves preservation of tissue morphology and dimensions. \u003cem\u003ePlant Methods\u003c/em\u003e, 9, 36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1746-4811-9-36\u003c/span\u003e\u003cspan address=\"10.1186/1746-4811-9-36\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson, R. H., \u0026amp; Wujek, D. E. (1997).Trentepohliales: Cephaleuros, Phycopeltis and related genera. Science Publishers.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUnited States Department of Agriculture (USDA). (2023). Oilseeds: World markets and trade. USDA Foreign Agricultural Service.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVorholt, J. A. (2012). Microbial life in the phyllosphere. Nature Reviews Microbiology, 10, 828\u0026ndash;840.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWidariyanto, R. Astari, S. \u0026amp; Sugandi, A. 2024. Morphological Characteristics and Identification of Algae Species on Oil Palm Leaves in an Oil Palm Estate in Riau, Indonesia. IOP Conference Series: Earth and Environmental Science, 1308(1): 012030.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuo, Y.-B., Han, D.-Y., Wang, Y.-Y., Yang, Q.-X., Ren, Q., Liu, X.-Z., \u0026amp; Wei, X.-L. (2023). Fungal\u0026ndash;Algal Association Drives Lichens\u0026rsquo; Mutualistic Symbiosis: A Case Study with Trebouxia-Related Lichens. Plants, 12(17), 3172\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Epiphytic Algae, Oil Palm, Phycopeltis arundinacea, Morphological Identification, Lichenisation","lastPublishedDoi":"10.21203/rs.3.rs-9540797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9540797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpiphytic red algae are commonly observed on oil palm leaves in humid tropical plantations; however, their taxonomic identity and ecological significance in Malaysia remain insufficiently documented. In other crops, similar algae are often associated with lichens, composite organisms formed through a symbiotic relationship between a photobiont and a mycobiont. Foliicolous lichens specifically develop on leaf surfaces of host plants. This study aimed to identify and characterise the epiphytic alga colonising oil palm (\u003cem\u003eElaeis guineensis\u003c/em\u003e) fronds, quantify symptom severity across frond ages and estates, and evaluate canopy-level spatial patterns. Leaf samples were collected from six oil palm estates in Peninsular Malaysia. Symptom severity was assessed using the Leaf Doctor digital image analysis application. Drone-acquired red, green, and blue imagery was analysed to compute the Visible Difference Vegetation Index. Morphological characterisation was conducted using stereomicroscopy, light microscopy, and scanning electron microscopy. Isolation trials were performed on Bold\u0026rsquo;s Basal Medium with and without Indole-3-Acetic Acid supplementation. Symptom severity increased significantly with frond age, with Fronds 25 and 33 exhibiting the highest colonisation levels (up to 84%). Vegetation index analysis revealed spatial clustering of affected areas. Microscopic observations confirmed discoid epiphytic thalli with radiating filaments and sporangial structures consistent with \u003cem\u003ePhycopeltis arundinacea\u003c/em\u003e as the dominant photobiont. Trebouxia-like cells and fungal associations were also observed, suggesting possible lichen-like interactions. The leaf epidermis remained intact, distinguishing this species from pathogenic \u003cem\u003eCephaleuros\u003c/em\u003e spp. This study provides the first confirmed report of \u003cem\u003ePhycopeltis arundinacea\u003c/em\u003e on oil palm in Malaysia and presents an integrated approach for epiphytic algal characterisation.\u003c/p\u003e","manuscriptTitle":"First Record of Phycopeltis Arundinacea (Trentepohliaceae, Chlorophyta) on Oil Palm (Elaeis Guineensis) in Malaysia: Morphological Characterisation and Evidence of Lichen-Associated Structures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 06:18:27","doi":"10.21203/rs.3.rs-9540797/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a12d8c26-d39b-49f1-9095-f3d911a0526d","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"checksComplete","content":"","date":"2026-05-19T09:44:32+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T06:18:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 06:18:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9540797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9540797","identity":"rs-9540797","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}