Bangladesh J. Plant Taxon. 32(1): 105-113, 2025 (June) DOI: https://doi.org/10.3329/bjpt.v32i1.82398 © 2025 Bangladesh Association of Plant Taxonomists MORPHO-MOLECULAR CHARACTERIZATION OF LASIODIPLODIA THEOBROMAE (PAT.) GRIFFON & MAUBL AND ITS FIRST REPORT ON THE ASSOCIATION WITH COCONUT KERNEL FROM BANGLADESH MD. ABDULLAH AL NOMAN 1, SHAMIM SHAMSI 1* AND ZEAUR RAHIM 2 1 Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh 2 International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh Keywords: Coconut; Lasiodiplodia isolate; Isolation; Characterization; Phylogenetic analysis. Abstract This study marks the first report of Lasiodiplodia theobromae associated with coconut in Bangladesh, a pathogen known to cause wide range of diseases crippling coconut production worldwide. Two isolates, Lt_BD 1 and Lt_BD 4, were obtained from coconut samples and subjected to comprehensive morpho-molecular and phylogenetic analyses. Morphological observations, including colony characteristics (color, texture, and surface appearance), growth patterns and conidial dimensions and shapes, preliminarily identified the isolates as Lasiodiplodia species. Molecular analysis, through PCR amplification of the internal transcribed spacer (ITS) regions, confirmed the identity of the isolates as L. theobromae. A phylogenetic tree, constructed using sequences of the studied isolates alongside 48 reference Lasiodiplodia species (retrieved from NCBI) and one out-group species (Pyricularia oryzae), corroborated this identification. This study provides a foundation for further rigorous research on the diseases of coconut caused by L. theobromae in Bangladesh. Introduction The coconut (Cocos nucifera L.), a member of the Arecaceae family, is one of the most vital perennial crops in tropical regions. Often termed the "tree of life," coconut offers diverse applications, ranging from food, oil, and medicine to construction materials, fibers, and cosmetics. The white flesh of the coconut is nutrient-rich, containing high levels of fats, carbohydrates, iron, potassium, vitamin A, and vitamin B. Its endosperm is widely consumed raw, used in confections, and forms the basis for various dishes and desserts. Coconut oil, extracted and processed from dried coconut, is good for skin and hair care. Economically, coconut is significant for Bangladesh, which exports pure and natural coconut products globally. Coconut production faces significant qualitative and quantitative challenges, with fungal diseases playing a critical role. Among these, phytopathogenic species from the genus Lasiodiplodia are responsible for approximately 500 plant diseases, including fruit rot, root rot, collar rot, stem-end rot, dieback, canker, and leaf necrosis (Huda-Shakirah et al., 2022). As a globally distributed pathogen, Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes, Ascomycota) affects a wide range of hosts and can exist as a parasite, saprophyte or endophyte in nature (Alves et al., 2008; Machado et al., 2014; Rosado et al., 2016). In coconut, Lasiodiplodia theobromae causes a wide range of diseases including nut fall (Venugopal and Mohanan, 2006; Sunpapao et al., 2022), leaf blight (Santos, 2020; Ramjegathesh et al., 2019; Ashokkumar et al., 2018), nut rot disease (Taylor and Hyde, 2003; Dheepa et al., 2018) and postharvest stem end rot (Rosado et al., 2016; Zhang and Niu, 2019), all of which cause serious hindrance to coconut production. Based on morphological, *Corresponding author. E-mail: botanyshamsi@du.ac.bd https://doi.org/10.3329/bjpt.v32i1.82398 106 NOMAN et al. phylogenetic and pathogenicity data, Santos et al. (2020) first addressed two species namely, Botryosphaeria fabicerciana and Lasiodiplodia pseudotheobromae in addition to L. theobromae as causal agents of leaf blight disease in coconut from Brazil. Previously, L. theobromae was the sole species linked to postharvest stem-end rot of coconut (Piepenbring, 2006; Taylor & Hyde, 2003). Rosado et al. (2016) expanded this understanding by reporting three additional species alongside L. theobromae namely, L. brasiliense, L. egyptiacae, and L. pseudotheobromae as causative agents of postharvest stem-end rot in Brazil. Their artificial inoculation experiments demonstrated that L. theobromae was the most prevalent and aggressive species causing the disease. Lasiodiplodia spp. are capable of surviving endophytically, enabling them to evade detection during quarantine. These fungi can infiltrate the endosperm, rendering coconut water unsuitable for consumption. Additionally, research by Felix et al. (2018) underscores the health risks posed by toxic metabolites produced by L. theobromae strains. Therefore, identification and proper characterization of this fungus in coconut is desperately needed. Several studies on fungal association with coconut have been reported from Bangladesh (Bhuiyan et al., 2021; Khan and Hossain, 2014). However, to the best of our knowledge, association of Lasiodiplodia theobromae with coconut has not yet been reported from Bangladesh. Therefore, this study aims to address this gap by characterizing L. theobromae isolates obtained from coconut kernels. Materials and Methods Sample collection and pathogen isolation Coconut fruit with characteristic symptoms was collected for isolation. The rotted coconut kernel was associated with dark brown to blackish mycelial patches which is a typical feature of fungi belonging to the Botryosphaeriaceae. Fungus was isolated directly from symptomatic fruits using the method described by Hosen et al. (2023). Details of isolate ID, origin, and corresponding NCBI accession numbers are provided in Table 1. The fungal isolates were incubated at 25°C for five days to obtain pure cultures, which were subsequently used for morphological and molecular characterization. For long-term preservation and future molecular analyses, the isolates were grown on sterile 3 mm filter paper disks and stored in sterile Eppendorf tubes at −80°C. Morphological identification Preliminary identification of fungal isolates was performed based on morphological characteristics, encompassing both macroscopic and microscopic features including conidia, conidiogenous cells and mycelium. Spore images were measured at 40x magnification using a Nikon Optiphot-2 trinocular microscope (Japan) equipped with a digital camera and ImageFocus Alpha software. For each isolate, the length and width of ten spores were recorded, and the average size was calculated. Molecular characterization and phylogenetic analysis DNA extraction Approximately 1 gram of mycelium from 7-day-old culture for each isolate was transferred into a 1.5 ml sterile Eppendorf tube. The mycelium was ground using a homogenizer in 400 μl of sterile extraction buffer (200 mM Tris-HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS). Genomic DNA was extracted following the protocol described by Noman et al. (2021). The resulting DNA pellet was resuspended in 100 μL of 1x TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and allowed to dissolve overnight at 4°C. The DNA samples were stored at −20°C for subsequent analyses. MORPHO-MOLECULAR CHARACTERIZATION OF LASIODIPLODIA THEOBROMAE 107 DNA concentration was measured at 260 nm using a Nanodrop spectrophotometer, and quality was assessed by electrophoresis on a 1% agarose gel prior to PCR amplification. Table 1. Details of the Lasiodiplodia theobromae isolates isolated from coconut kernel during the present study and the reference isolates retrieved from NCBI for phylogenetic analysis. Sl. No. a,b&c Name of the isolate Isolate ID Origin of the isolate NCBI accession no. References Location Host 1. Lasiodiplodia theobromae Lt_BD 1 Bangladesh Cocos nucifera OQ438652 This study 2. L. theobromae Lt_BD 4 Bangladesh C. nucifera OQ438653 This study 3. L. theobromae PCB Malaysia Jatropha curcas GU228527 Sulaiman et al., 2012 4. L. theobromae RSGV/LK02 Malaysia J. curcas HM346873 Sulaiman et al., 2012 5. L. theobromae FH14K03 Mexico Citrus tree MK886711 Hernández et al., 2021 6. L. theobromae Lt1 India Bottle Gourd MN995068 Unpublished Jain S and Singh G 7. L. theobromae 1_finish China Poplar stem KF294005 Unpublished Sun XM and Yan DH 8. L. theobromae YLH2-2 China Avocado OM736159 Unpublished Yu HR and Wu JB 9. L. pyriformis CBS 121770 Namibia Acacia mellifera EU101307 Cruywagen et al., 2017 10. L. pyriformis CMW 25415 Namibia A. mellifera EU101308 Cruywagen et al., 2017 11. L. egyptiacae BOT-29 Egypt Mangifera indica JN814401 Ismail et al., 2012 12. L. egyptiacae BOT-10 Egypt M. indica JN814397 Ismail et al., 2012 13. L. subglobosa CMM 3872 Brazil Jatropha curcas KF234558 Gnanesh et al., 2022 14. L. subglobosa CMM 4046 Brazil J. curcas KF234560 Gnanesh et al., 2022 15. L. gilanensis IRAN1501C Iran Unknown GU945352 Abdollahzadeh et al., 2010 16. L. gilanensis IRAN1523C Iran Unknown GU945351 Abdollahzadeh et al., 2010 17. L. venezuelensis WAC12539 Venezuela Acacia mangium DQ103547 Burgess et al., 2006 18. L. venezuelensis WAC12540 Venezuela A. mangium DQ103548 Burgess et al., 2006 19. L. venezuelensis CMW 13513 Venezuela A. mangium DQ103549 Burgess et al., 2006 20. L. rubropurpurea WAC12535 Tully, Queensland Eucalyptus grandis DQ103553 Burgess et al., 2006 21. L. rubropurpurea WAC12536 Tully, Queensland E. grandis DQ103554 Burgess et al., 2006 22. L. rubropurpurea WAC12537 Tully, Queensland E. grandis DQ103555 Burgess et al., 2006 23. L. rubropurpurea WAC12538 Tully, Queensland E. grandis DQ103556 Burgess et al., 2006 24. L. citricola CBS124707a Iran Citrus sp. GU945354 Abdollahzadeh et al., 2010 25. L. citricola CBS124706 Iran Citrus sp. GU945353 Abdollahzadeh et al., 2010 26. L. crassispora CBS125626 South Africa Vitis vinifera MT587424 Zhang et al., 2021 27. L. crassispora CMW33262 Unknown Adansonia sp. KU887068 Cruywagen et al., 2017 28. L. crassispora CMW 13488 Venezuela Eucalyptus europhylla DQ103552 Gnanesh et al., 2022 https://www.ncbi.nlm.nih.gov/nuccore/OQ438652 https://www.ncbi.nlm.nih.gov/nuccore/OQ438653 108 NOMAN et al. 29. L. crassispora CBS 118741 Australia Santalum album NG_062741 Phillips et al., 2005 30. L. euphorbicola CMM3651 Brazil Jatropha curcas KF234553 Machado et al., 2014 31. L. euphorbicola CMW33268 Unknown Adansonia sp. KU887131 Cruywagen et al., 2017 32. L. euphorbicola CMM3609 Brasil Jatropha curcas KF254926 Machado et al., 2014 33. L. mahajangana CBS124925 Madagascar Terminalia catappa FJ900595 Begoude et al., 2010 34. L. mahajangana CBS124926 Madagascar T. catappa FJ900596 Begoude et al., 2010 35. L. hormozganensis CBS124709 Iran Olea sp. GU945355 Abdollahzadeh et al., 2010 36. L. hormozganensis CBS124708 Iran Mangifera indica GU945356 Abdollahzadeh et al., 2010 37. L. margaritacea CBS122519 Australia Adansonia gibbosa EU144050 Cruywagen et al., 2017 38. L. margaritacea CBS122065 Australia A. gibbosa EU144051 Cruywagen et al., 2017 39. L. margaritacea CBS138289 Namibia Combretum elaeagnoides KP872320 Zhang et al., 2021 40. L. margaritacea CBS138290 Zambia Combretum collinum KP872321 Zhang et al., 2021 41. L. parva CBS 356.59 Sri Lanka Theobromae cacao EF622082 Ismail et al., 2012 42. L. parva CBS 494.78 Colombia Cassava-field soil EF622084 Ismail et al., 2012 43. L. exigua BL 184 Tunisia Retama raetam KJ638318 Linaldeddu et al., 2015 44. L. exigua BL 185 Tunisia R. raetam KJ638319 Linaldeddu et al., 2015 45. L. exigua BL 187 Tunisia R. raetam KJ638321 Linaldeddu et al., 2015 46. L. exigua CBS 137785 Tunisia R. raetam KJ638317 Linaldeddu et al., 2015 47. L. brasiliense CBS123095 Cameroon Teobroma cacao MT587423 Zhang et al., 2021 48. L. brasiliense CMM4015a Brazil Mangifera indica JX464063 Marques et al., 2013 49. L. brasiliense CSM11 Venezuela Teobroma cacao MF436018 Mohali-Castillo et al., 2023 50. L. brasiliense CF/UENF436 Brazil Cocos nucifera KY655209 Santos et al., 2020 51. Pyricularia oryzae BDC_10 Bangladesh Triticum aestivum MT358609 Noman et al., 2021 a Lasiodiplodia theobromae isolates studied in the present investigation are shown in bold (1&2) b Reference Lasiodiplodia isolates obtained from NCBI and used for phylogenetic analysis (03-50) c Reference Pyricularia oryzae isolate obtained from NCBI and used as out-group for phylogenetic analysis (51) PCR amplification and sequencing The internal transcribed spacer (ITS) regions of the isolates were amplified using the forward primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and the reverse primer ITS4 (5'- TCCTCCGCTTATTGATATGC-3') (White et al., 1990). Each 25 μl PCR reaction mixture contained 2.0 μl of template DNA, 12.5 μl of Master Mix (Clever Scientific Ltd., Warwickshire, UK), 1.0 μl of each primer, and 8.5 μl of nuclease-free water. The reaction mixture was thoroughly mixed before thermal cycling, which included an initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 30 seconds. A final extension step at 72°C for 5 minutes was included, ending with a hold at 4°C. Successful amplification of the ITS regions was verified by electrophoresis on a 1% agarose gel with compared to a 100 bp DNA ladder (Clever Scientific Ltd., Warwickshire, UK). Purified PCR products were sequenced using a SeqStudio Genetic MORPHO-MOLECULAR CHARACTERIZATION OF LASIODIPLODIA THEOBROMAE 109 Analyzer (Thermo Fisher Scientific, USA) at the Centre for Advanced Research in Sciences (CARS), University of Dhaka, Bangladesh. Sequence analysis and phylogenetic tree construction The nucleotide homogeneity of the obtained consensus sequences was evaluated by comparing them with other sequences in the GenBank database using the BLASTn tool (http://www.ncbi.nlm.nih.gov/BLAST) and these sequences were subsequently deposited in the GenBank database. Sequence alignment was performed using the CLUSTAL W algorithm implemented in Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 (Kumar et al., 2016). A phylogenetic tree was constructed using the neighbor-joining method within the same software, and branch support was evaluated using 1000 bootstrap replicates. Results and Discussion Morphological characterization Morphological characteristics such as colony color and texture, surface appearance, growth pattern, and conidial size and shape were examined. The isolates grown on culture media displayed typical Lasiodiplodia morphology. The mycelium grew vigorously in all directions, completely covering the surface of the Petri plates within 5 days (Fig. 1). The colony texture of the Lasiodiplodia isolates was fluffy, raised and irregular. Initially, the colonies were white, gradually changing to light gray within a week. After two weeks of incubation, the color turned dark gray or black when viewed from the top and dark olive green or black from the reverse side (Fig. 1). No variation in conidial shape was observed. The conidia were septate, oval in shape, dark brown in color with irregular longitudinal striations on the spores. Average conidial sizes of the isolates Lt_BD 1 and Lt_BD 4 were found 22.5 × 12.0 µm and 23.0 × 11.5 µm, respectively. Fig. 1. Morphological characterization of L. theobromae. A. Infested coconut kurnel; B-C. 5 days old colony on PDA medium from upper (B) and reverse view (C); D-E. 14 days old mature colony on PDA medium from upper (D) and reverse view (E); F. Conidia under microscope (scale bar = 50 µm). 110 NOMAN et al. Molecular characterization and phylogenetic analysis Molecular identification and phylogenetic analysis were conducted to accurately identify the isolates at the species level. PCR amplification of the internal transcribed spacer (ITS) regions produced an amplicon of approximately 550 bp for each isolate (Fig. 2). The amplicons were purified, sequenced, and analyzed using the NCBI BLAST search tool. The ITS sequences of the isolates were found to be identical and confirmed as L. theobromae. The newly generated sequences from this study were submitted to NCBI, and the corresponding GenBank accession numbers are listed in Table 1. Fig. 2. Gel electrophoresis of amplified ITS region of the L. theobromae isolates using 1% agarose gel (M indicates 100 bp DNA ladder). To analyze the phylogenetic position of the studied L. theobromae isolates, a neighbor-joining tree was also constructed based on ITS sequences. The ITS sequences of the isolates from the present study were aligned with 48 reference isolates of Lasiodiplodia species of different countries and plant hosts (retrieved from NCBI) and one outgroup taxon (Pyricularia oryzae) (Fig. 3). From the phylogenetic tree it was observed that out-group taxon, P. oryzae, clustered completely separately and remaining all the Lasiodiplodia species formed a major cluster among them. Species wise clustering was demonstrated in the dendrogram. Isolates of this study namely, Lt_BD 1 and Lt_BD 4 showed strong relationship with reference L. theobromae isolates and formed a different cluster. As a result, isolates of this study were verified as L. theobromae by virtue of molecular identification and phylogenetic analysis. Lasiodiplodia theobromae has previously been identified as a pathogen of dragon fruit in Bangladesh (Briste et al., 2021). However, despite being serious pathogen of coconut, there is no available report on the association of this fungus with coconut from Bangladesh till date. This study marks the association of L. theobromae with coconut from Bangladesh and demonstrated detailed morpho-molecular characterization with phylogenetic relationship. The isolates studied here are preliminarily identified as Lasiodiplodia species based on their morphological features, consistent with descriptions provided by other researchers studying Lasiodiplodia (Alves et al., 2008; Marques et al., 2013; Machado et al., 2014; Linaldeddu et al., 2015; Rosado et al., 2016; Huda-Shakirah et al., 2022). Morphological methods have traditionally been central to fungal taxonomy. However, morphology-based identification within the Botryosphaeriaceae family is limited to the genus level, as many Lasiodiplodia species share overlapping morphological traits. This limitation highlights the importance of molecular techniques. As a result, molecular and phylogenetic investigations incorporating ITS DNA sequences are critical for avoiding ambiguous and misleading results and resolving species-level identification issues. ITS region is recognized as a MORPHO-MOLECULAR CHARACTERIZATION OF LASIODIPLODIA THEOBROMAE 111 universal fungal barcode and an effective molecular tool for identifying fungal species and analyzing the phylogenetic relationships of various species and geographic isolates (Rosado et al., 2016; Noman et al., 2021). In this study, molecular characterization of the isolates was carried out using rDNA sequences of the ITS region. Neighbor-joining tree inferred from L. theobromae isolates of this study together with 48 reference Lasiodiplodia species and one outgroup taxon (Pyricularia oryzae) revealed that studied Lt_BD 1 and Lt_BD 4 isolates and other reference L. theobromae isolates showed strong relationship and formed a completely separate cluster, confirming that studied fungal isolates were L. theobromae. Fig. 3. Phylogenetic relationship of Lasiodiplodia theobromae isolates with other reference Lasiodiplodia isolates based on ITS sequence similarity using neighbour- joining method. L. theobromae isolates of this study, reference L. theobromae isolates and reference Pyricularia oryzae isolate (out-group) were marked in green, yellow and light orange zones, respectively. Numbers besides each branch represent bootstrap values obtained after a bootstrap test with 1000 replications. Branch support less than 40 was not shown in the dendrogram. 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