Isolation and Identification of Endophytic Fungi from the Roots of two epiphytic orchids: Rhynchostylis retusa (L.) Bl. and Vanda tessellata (L.) Bl

Isolation and Identification of Endophytic Fungi from the Roots of two epiphytic orchids: Rhynchostylis retusa (L.) Bl. and Vanda tessellata (L.) 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Bl. and Vanda tessellata (L.) Bl M Abu Sayem, M Shahin Miah, Mohammad Musharof Hossain This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4463026/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 Two fungal endophytes were isolated and identified from two indigenous orchids of Bangladesh namely, Rhynchostylis retusa (L.) Bl. and Vanda tessellata (L.) Bl. Nature of fungal colonization, seasonal variations of colonization in root cortex cells were also studied. The fungal endophytes isolated from two different orchids differed in their cultural morphology and microscopic features such as colony morphology, colour of the colony, presence/absence of monilioid cells or spores and diameter of vegetative hyphae. The microscopic features i.e ., hyphal structure, right-angle branching of hyphae, slight constriction at the branching point, shape and diameter of monilioid cells of the endophytic fungus isolated from R. retusa showed resemblance with the anamorphic Rhizoctonia -like fungi Ceratobasidium sp. On the other hand, the fungal endophytes isolated from the roots of V. tessellata produced huge number of spores. The cultural characteristics and spore morphology of this fungi corroborated resemblance with pathogenic fungi, Fusarium sp. The identity of the fungi was further reconfirmed through sequencing of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA (nrDNA). The BLASTn search of ITS region sequences of the endophytic fungi isolated from R. retusa exhibited maximum similarity (97%) with an orchid mycorrhizal fungi (OMF), Ceratobasidium papillatum (GeneBank Accession No. OQ073691), while the fungal endophyte isolated from V. tessellata showed maximum similarity (99%) with Fusarium ambrosium (GeneBank Accession No. OP752102). The phylogenetic tree constructed using ITS region sequences of the isolated fungal endophytes and their closely linked species from genebank data developed two distinct groups. The conventional and molecular approaches applied for identification of these OMF can be followed for easy and accurate identification of other OMF. Orchid mycorrhiza Epiphytic orchid ITS sequencing Fusarium Ceratobasidium Figures Figure 1 Figure 2 Figure 3 Introduction Under natural or horticultural environments, an archetypal mutualistic association is established among certain heterogeneous group of fungi and orchid roots termed as orchid mycorrhizae (OM). The term mycorrhiza (Fungus Root) was first coined by Professor A. B. Frank in 1885. Systematic studies on OM started more than a century ago and established the fundamental fact that mycorrhizal association is universal in the Orchidaceae, it facilitates nutrients assimilation, seed germination, and act as biological barriers against pathogens (Harris-Valle et al. 2021 ; Hossain 2022 ). No ̈el Bernard judiciously observed OM and hypothesized that fungus and hosts are nutritionally reliant to each other (Selosse et al. 2011 ). Frank hypothesized that orchid mycorrhizae represent a pervasive mutualistic symbiosis where fungus extracts mineral nutrients from surrounding environment and translocate them to their host plants; and in turn the host plants give shelter and nourish the fungi (Yam et al. 2009). Mycorrhizas are classified under 7 groups i.e ., Arbuscular mycorrhiza (AM), Arbutoid mycorrhiza, Ectomycorrhiza (EM), Ericoid mycorrhiza, Monotropid mycorrhiza, Ectendo mycorrhiza and Orchid mycorrhiza (Bellgard et al. 2011). Orchid mycorrhiza is very unique in their structure and functions. Mycorrhizal endophytes colonize in the cortex cells of orchid roots and develop a dense coil like structure by fungal hyphae are known as "peloton". Inside the host cells the pelotons are bounded by a membrane of interfacial matrix material. The difference between this membrane and plasma membrane is that this surrounding membrane lacks adenylate cyclase activity. The alignment of cell wall microfibrils and microtubules is changed during infection and may be essential for alteration in the cytoplasm and synthesis of the surrounding membrane of the pelotons. The activity of ascorbic acid oxidase, peroxidase, catalase and polyphenol oxidase increased mycorrhizal infection and help in pelotons digestion in the host cells. Even with their dependency on these mycorrhizal fungi, orchids can control the level of mycorrhizal infection. They regulate fungal growth through the release of a specialized phytotoxin ‘Orchinol’ which is believed to inhibit fungal growth (Hadley 1982 ). Some other literatures suggest that the mature pelotons are digested as a result of the activity of the lytic enzyme ‘orchinol’ (phytoalexins) produced by the host and release nutrients for host plants. Once the mycorrhizal symbiosis established between orchid roots and fungi, the fungi served as dominant microorganisms and release antagonistic chemical substances, effective in controlling the invasion of other pathogens and enhance growth and survival of seedlings (Hossain et al. 2013 ; Hossain 2022 ). The fungus provides carbohydrates, vitamins and growth stimulus that meet up the nutrient demand of germinating seeds. Fungal exudates stimulate the growth rate as well as the germination percentage in vitro . Symbiotic seed germination of terrestrial orchids has been used as an alternative to asymbiotic methods with success (Rasmussen 1995 , 2002 ; Johnson et al. 2007 ; Jolman et al. 2022 ). Mycorrhizal fungi provide the basic organic carbon and carbohydrates for seedling growth. Adult individuals of achlorophyllous and photosynthetic orchids obtain part of carbon heterotrophically by mycorrhizal fungi (Bidartondo et al. 2004 ; Julou et al. 2005 ; Selosse et al. 2009; Jacquemyn et al. 2010 ). Orchids also receive some organic compounds other than carbon from their fungal partners. Goodyera repens associated with mycorrhizal fungi acquired 100 times more phosphorus than non-mycorrhizal controls (Alexander et al. 1984 ). Phosphorus and Nitrogen (as glycine) transfer from fungus to plant was confirmed through radio labelling experiments (Cameron et al. 2006 ). Mycorrhizal fungi may also be a key source of water for orchids. In both the terrestrial Platanthera integrilabia and the epiphytic Epidendrum conopseum water content was higher for mycorrhizal seedlings than uncolonized controls (Yoder et al., 2000 ). In adult photosynthetic orchids nitrogen. phosphorus and water continue to flow from the fungal partner but carbon exchange is essentially reversed with photosynthetic providing incentive for continued fungal colonization. Endophytic fungal associations increase host plant fitness to abiotic stresses (Redman et al. 2002 ; Bae et al. 2008 ) and improve plant adaptability to various environmental conditions (Bonnardeaux et al. 2007 ; Swarts et al. 2010 ). From the beginning of orchid mycorrhizal research, it was believed that Rhizoctonia is the only fungal partner associated with orchids. Afterward, a number of diverse fungi were isolated and identified from different orchids which commonly termed as Rhizoctonia -like fungi because they exhibit similarity in many characteristics to Rhizoctonia (Bhuiyan et al. 2021 ; Hossain 2022 ). Recently orchid mycorrhizal fungi are using for biohardening of in vitro grown plantlets to enhance survival rates to ex vitro environment (Hossain et al. 2013 , 2022). They also have remarkable role in enhancing both reproductive and vegetative growth of orchid plants; stimulate early flowering, improve flower quality; and reduce severity if disease infection (Hossain 2022 ). The aim of the present research program is to isolate and identify OMF from two important indigenous orchids of Bangladesh namely, Rhynchostylis retusa and Venda tesselata . Materials and Methods Fresh mycorrhizal roots of Vanda tessellata and Rhynchostylis retusa were collected in air-tight plastic bags from the naturally grown plants during November-December (winter season) and May-June (rainy season) and used for isolation of fungal endophytes within 24 hrs of collection. The root samples were washed under running tap water to remove dust particles and debrides from root surface and cut into thin transverse sections at different portions of root and observed under microscope by staining with lactophenol cotton blue to confirm the presence of fungal pelotons. The roots showed the occurrence of fungal pelotons were used for isolation of fungal endophytes. The occurrence of fungal endophytes and formation of pelotons in the root cortex cells were calculated by following formula (Hossain 2019 ): % fungal colonized cells in the root cortex $$=\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{e}\text{l}\text{l}\text{s} \text{s}\text{h}\text{o}\text{w}\text{i}\text{n}\text{g} \text{c}\text{o}\text{l}\text{o}\text{n}\text{i}\text{z}\text{a}\text{t}\text{i}\text{o}\text{n} \text{p}\text{e}\text{r} \text{m}\text{i}\text{c}\text{r}\text{o}\text{s}\text{c}\text{o}\text{p}\text{i}\text{c} \text{v}\text{i}\text{e}\text{w} }{\text{T}\text{o}\text{t}\text{a}\text{l} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{e}\text{l}\text{l}\text{s} \text{p}\text{e}\text{r} \text{m}\text{i}\text{c}\text{r}\text{o}\text{s}\text{c}\text{o}\text{p}\text{i}\text{c} \text{v}\text{i}\text{e}\text{w}}\times 100\%$$ The roots showed the occurrence of fungal pelotons were washed with distilled water and treated by 0.1% HgCl 2 for 6 to 10 mins for surface sterilization. Afterword these were washed with sterile distilled water for 3 times to remove mercuric chloride solution. Final disinfection of the roots was completed by dipping in 70% ethanol for 30 sec to 1 min and washed thoroughly 3 times by double sterile distilled water. The sterilized mycorrhizal roots were then aseptically cut transverse sections approximately 1–2 mm thickness and cultured in Petri dishes containing potato dextrose agar (PDA) medium. The culture plates were incubated at 27 ± 2 °C in an incubator until fungi were grown visibly from root sections onto the PDA surface. The fungi that only grown from the inner portion of the cultured root sections were taken in consideration as possible mycorrhizal fungi. Pure fungal cultures were achieved through transferring a bit of hyphal tips from actively growing fungal colony onto fresh PDA medium. Nature of fungal colony growth, both surface and reverse colours of the colony at young and mature stages were recorded. The diameter of the hyphae and dimensions of spores or monilioid cells were measured by light microscope (Nikon E600, Tokyo, Japan) through mounting the mycelium with lactophenol cotton blue taken on glass slides. Colony growth rates were measured according to Currah et al. ( 1987 ) by inoculating uniform mycelial bits at the middle of petri plates containing PDA medium and represented by averages based on three replications. Radial increments in colony size were measured at 48 hrs interval over two weeks. For determining nuclei number per vegetative cell, spore or monilioid cell; a small part of the mycelial mat was taken on a glass slide, fixed in 2% formaldehyde for 2 min and cleaned with sterile distilled water for 1 min, followed by staining for 10 min with gold antifade diamidino-2-phenylindole (DAPI, ProLong®, Invitrogen Ltd., Eugene, USA), and destained with sterile distilled water for 2 min. Thereafter, a drop of 50% glycerin was spread over the DAPI stained sample, protected by cover slip and observed under microscope equipped with fluorescence accessory with mercury lamp. Micrographs of different fungal parts were taken by Nikon E600 microscope furnished with photographic accessories. For extraction of genomic DNA 1g mycelial mat was taken from two weeks old fungal cultures following the protocol described by Liu et al. ( 2000 ). The ITS1, 5.8S ribosomal RNA gene and ITS2 were amplified using forward and reverse primers ITS1 (5’ TCCGTAGGTGAACCTGCGG) and ITS4 primers (5’GCTGCGTTCATCGATGC) (White et al. 1990 ). The PCR master mix was prepared in 50 µl reaction matrix containing 2 µl of genomic DNA, 5 µl of dNTPs mixture, 1 µl of each primer (10 pmol), 5 µl of 10× PCR buffer, 0.4 µl of Taq polymerase 1U, and 35.6 µl of MiliQ water. The PCR cycle condition was an initial denaturation at 94°C for 5 min following 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 45 sec and final extension at 72°C for 7 min. The PCR amplicons were purified, concentrated and generated the nucleotide sequences of the PCR products by a DNA Analyzer (Thermo Fisher Scientific, McLab, California, USA). A consensus sequence was generated from the forward and reverse sequences by using BioEdit 7.2.6 software (Hall, 1999 ). The consensus sequence was deposited in the NCBI GenBank (Accession no. OQ073691 and OP752102). The consensus sequence was placed into web based Basic Local Alignment Search Tool (BLAST) of NCBI GenBank ( https://blast.ncbi.nlm.nih.gov/ ) to find out similar species/isolates. The phylogenetic relationships were established based on maximum parsimony tree by using analysis program of the MEGA version 11.0.13 software (Tamura et al., 2021 ). In this analysis Bootstrap replications number was 1000. Results and Discussion Two fungal endophytes, one from each orchid species, were isolated from the mature roots of the epiphytic orchids namely, Rhynchostylis retusa (L.) Bl. and Vanda tessellata (L.) Bl. The fungal endophytes showed intra- and inter-cellular colonization of the cortical cells of naturally grown plants with pelotons formation within the matured roots of the selected orchids. Fungal colonization was detected only in the root portions attached to the substratum in both V. tessellata (Fig. 1 A) and R. retusa (Fig. 2 A). The mycorrhizal colonization in the selected orchids were occupied a considerable proportion of the cortex cells. No pelotons were found in the root tip regions in both cases. The pelotons appeared in the colonized roots as loose coils surrounded by live hyphae in the outer-cortex considered as young pelotons and brownish in the inner cortex which considered as old pelotons. The percentage of fungal colonization differed in different growing seasons. The incidence of root colonization by fungi was significantly higher in rainy season (June-July) than dry season (November-December) in both the orchid species. In case of R. retusa , the root cortex cells colonization percentage was 95.65 ± 1.99% in rainy season and 76.52 ± 1.22% in winter seasons while in the roots of V. tessellata showed 88.10 ± 1.55% cell colonization in rainy season and 65.14 ± 1.07% in winter season. The percentage of fungal colonization in orchid roots also varies according to the root types. Bertolini et al. ( 2014 ) demonstrated that mycorrhization is meticulously extant in orchid roots and it is dependent on the wet and dry seasons. They also checked that rainfall determines higher fungal colonization and the presence of greater number of fresh and undigested live pelotons in the roots. Similar findings were also reported in Gastrochilus calceolaris (Hossain 2019 ) and R. retusa (Bhuiyan et al. 2021 ) and Venda tesselata (Shagufta et al. 1993 ). The greater intensity of mycorrhizal colonization in rainy season and thoroughgoing occurrence of pelotons in root portions that directly contact with the substrate is reported in epiphytic orchids, Epidendrum stamfordianum , Erycina crista-galli , Stelis quadrifida (Bertolini et al. 2014 ). Reasons behind maximum cell colonization by fungi during summer and rainy seasons for vigorous vegetative growth and flowering of plant as compared to the winter season of slow growth and dry weather. These observations validated the previous reports of higher colonization by well-matched fungi to meet up the additional nutrient demand for vigorous growth and flowering of orchids (Hossain 2019 , 2022 ; Buiyan et al. 2021). Mycorrhizal fungi are sensitive to environmental conditions and seasonal effects influence the structures of mycorrhizal association in the orchid (Han et al. 2016 ). They also noticed that the decrease of fungal population density and change the leading associative fungi across the two different seasons; for example, the common OMF Tulasnella and Russula significantly reduced their abundance in the dry season. The pelotons in the inner-cortex displayed brownish coloured loose hyphal coils as a result of breakdown of fungal hyphae by the activity of lytic enzymes produced by the host cells and discharge nutrients. Orchids also show sensible defense mechanisms for controlling the level of fungal penetration and colonization by producing phytoalexins or orchinol. Colony growth appearance of fungi, colour of the young and mature colony in both front and reverse view and growth rate were studied. In the case of fungi isolated from R. retusa , cottony growth of the colony was observed. The young colonies were white colour at the front side (Fig. 1 B) and off-white colour at the reverse side. The old colonies were greyish on the front side and brown at the back side (Fig. 1 C,D). In the case of the fungi isolated from V. tessellata , cottony appearance of the fungal colony was found. The young colonies were white at the front side (Fig. 2 B) and greyish white at the back side. The old colonies were slightly brownish at the front side (Fig. 2 C) and yellowish at the reverse side. The average colony growth rate of the fungi isolated from R. retusa was 0.26 mm/h while in the fungi isolated from V. tessellata was 0.30 mm/h. The microscopic features of the fungal endophytes i.e . hyphal structure, hyphal diameter, shape, size and diameter of monilioid cells, branching pattern, colour of the vegetative hyphae of the fungal endophyte isolated from R. retusa exhibited resemblance with the anamorphic Rhizoctonia -like fungus Ceratobasidium sp. The monilioid cells were elipsoidal and in chain form with slightly thicker wall than the vegetative hyphae (Fig. 1 E). The endophytes showed a slight constriction at each hyphal branching point with a dolipore septum subtending the hyphal branch slightly above the constriction (Fig. 1 F). In case of the fungus isolated from the roots of V. tessellata , the colour of the hyphae was also hyaline. The hyphae were less than 5µm in diameter (Fig. 2 D). This isolate had no special hyphal character as like the fungi isolated from R. retusa . Monilioid cells were absent but huge number of round shaped microspores (Fig. 2 E), multicellular macrospores (Fig. 2 F) and round shape chlamydospores were produced. The spore morphology showed resemblance to Fusarium sp. Other than mycorrhiza forming fungi, there are many endophytic and surface associated fungi were documented from orchid roots. These associated fungi are especially abundant in terrestrial orchid roots. The non-mycorrhizal orchid endophytic fungi are more diverse than Rhizoctonia -like fungi which comprise over 110 genera, for example, Fusarium, Trichoderma, Armillaria, Cochliobolus, Sclerotiana , Merismodes, Hypocrea , etc. (Ma et al. 2015 ). The role of non-mycorrhizal orchid endophytes is comparatively less explored. It is presumed that non-mycorrhizal endophytes play a vital role in growth and development of orchids. Fungal endophytes such as Alternaria sp., Fusarium oxysporum isolated from terrestrial orchids in Brazil (Vaz et al. 2009 ). From the beginning of orchid mycorrhizal study, only Rhizoctonia -like fungi i.e . Ceratobasidium , Thanatephorus , Tulasnella , Sebacina, Mycena etc. was considered OMF. Subsequently, some other fungal species such as Trichoderma spp., Fusarium sp., Aspergillus spp., Piriformospora sp., Serendipita sp., Gymnopus sp. have been isolated from orchids as root endophytes (Hossain 2022 ). Very recently, Fusarium sp. has been reported as a potential and common endophytic fungus from different orchids including Venda tesselata (Rasmussen et al. 2001; Behera et al. 2013 , Jiang et al. 2019 ; Fuji et al. 2020 ; Hernández-Martínez et al. 2020 ; Tian et al. 2022 ). The morphological identity of the above fungal endophytes was further confirmed through molecular techniques by sequencing and analysis of Internal Transcribed Spacer (ITS) sequences of the nuclear ribosomal DNA (nrDNA) of the endophytes. The BLASTn search of ITS region sequences of the fungal endophytes isolated from R. retusa showed maximum similarity (97%) with Ceratobasidium papillatum (GeneBank Accession No. OQ073691), while the fungal endophytes isolated from V. tesselata exhibited maximum resemblance (99%) with Fusarium ambrosium (GeneBank Accession No. OP752102). The phylogenetic tree based on ITS region sequences of the fungal endophytes and their closely related species formed two distinct groups (Fig. 3 ). The ITS region sequencing is a widely used and powerful molecular technique for precise identification of OMF (Sharon et al. 2008 ; Hossain 2022 ). This region is considered as a universal ‘barcode’ for fungal identification because it has some special features that make it a strong candidate for barcoding. For example, it is easy to amplify because of its high copy number, comparatively few primer sets are required, it has highly conserved small sub-unit and large sub-unit neighboring regions, and varies dramatically between species but relatively little within species, and far better represented than other loci in fungi in the GenBank (Taylor and McCormick 2008 ; Zettler and Corey 2018 ; Hossain 2022 ). Therefore, molecular characterization validated the morphological information. This study also provides new information about Fusarium as root associated fungi in orchids and the techniques adapted for this fungus will facilitate to isolate and precise identification of other orchids mycorrhizal fungi. Declarations Conflict of interests The authors declare no conflict of interests. Author Contribution MM Hossain made substantial contributions to the conception and design of the study and drafted the manuscript; MA Sayem and MS Miah completed the experiment and contributed to writing the manuscript. All authors approved the draft of the manuscript. Acknowledgements The authors gratefully acknowledge the ‘Research and Publication Cell’ of the Chittagong University, Bangladesh for providing financial support. References Alexander C, Alexander IJ, Hadley G (1984) Phosphate uptake by Goodyera repens in relation to mycorrhizal infection. 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In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR protocols: A guide to methods and applications, Academic Press, San Diego, California, USA. pp 315-322 Yam TW, Arditti J (2009) History of orchid propagation: A mirror of the history of biotechnology. Plant Biotechnol Rep 3:1–56 Yoder JA, Zettler LW, Stewart SL (2000) Water requirements of terrestrial and epiphytic orchid seeds and seedlings, and evidence for water uptake by means of mycotrophy. Plant Sci 156:145-150 Yuan ZL, Chen YC, Yang Y (2009) Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic, medicinal orchid ( Dendrobium nobile ): Estimation and characterization. World J Microb Biotech 25(2):295-303 Zettler LW, Corey LL (2018) Orchid mycorrhizal fungi: isolation and identification techniques. In: Orchid propagation: from laboratories to greenhouses—methods and protocols. Humana Press, New York, NY. pp 27-59 Additional Declarations No competing interests reported. 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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-4463026","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309700367,"identity":"f117af66-cc3a-4b74-9455-13b38ba9cfc7","order_by":0,"name":"M Abu Sayem","email":"","orcid":"","institution":"University of Chittagong","correspondingAuthor":false,"prefix":"","firstName":"M","middleName":"Abu","lastName":"Sayem","suffix":""},{"id":309700368,"identity":"2b33f948-2c20-408a-aa9e-38626b219704","order_by":1,"name":"M Shahin Miah","email":"","orcid":"","institution":"University of Chittagong","correspondingAuthor":false,"prefix":"","firstName":"M","middleName":"Shahin","lastName":"Miah","suffix":""},{"id":309700369,"identity":"90347aa3-126e-4016-95d2-507188832353","order_by":2,"name":"Mohammad Musharof Hossain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYHACNhDBz8/MwyDxgYEhgWgtkjPbexgkZ5CkZcOZMwzSPMRo0Z2R/uzBzz02Egw3cg/etm2zy+Nnb2D88DEHtxazGznmhj3P0iQYZ+QlW+e2JRdL9hxglpy5Da8WNgmeA4frmCVyzKRz25gTN9xIYGPmxasl/ZnknwOHJdhAWizb6onRkmAmDbRFgofnjJk0Y9thIrSceWMmLXMgTUKCvS/Zsufc8cSZPQeb8fvlONBhbw7YSNgf5j1440dZdWI/e/PBDx/xaEEFjOA4YmwgVj0I/CFF8SgYBaNgFIwUAACuQlPf9q5FVAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Chittagong","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"Musharof","lastName":"Hossain","suffix":""}],"badges":[],"createdAt":"2024-05-22 20:46:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4463026/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4463026/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57749461,"identity":"82cf57fc-5e19-4455-9013-1a273f718a49","added_by":"auto","created_at":"2024-06-05 06:41:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":216462,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fungal pelotons in the roots of \u003cem\u003eR. retusa\u003c/em\u003e, (B) young fungal colony (front side) growing on PDA surface, (C) two week old mature colony of fungal endophyte (front side), (D) reverse view of the same culture, (E) elipsoidal monilioid cells, and (F) Fungal hyphae \u0026nbsp;with slight constriction at hyphal branching point and dolipore septum subtending the hyphal branch.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4463026/v1/fac767387cbe5bd209998f93.jpg"},{"id":57749463,"identity":"a216046c-4643-4eb9-abd4-9f758ae092a0","added_by":"auto","created_at":"2024-06-05 06:41:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226155,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fungal pelotons in the roots of \u003cem\u003eV. tessellata\u003c/em\u003e, (B) young fungal colony (front side) growing on PDA surface, (C) two week old mature colony of fungal endophyte (front side), (D) branching pattern of hyphae and nuclear number per vegitative cell, (E) round shaped microspores, and (F) multicellular macrospores.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4463026/v1/86d74e6f009661cafdd02369.jpg"},{"id":57749462,"identity":"a35257de-b607-42bc-8736-35aacd0fa518","added_by":"auto","created_at":"2024-06-05 06:41:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":147220,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on ITS region sequences of the fungal endophytes isolated from \u003cem\u003eR. retusa\u003c/em\u003e and \u003cem\u003eV. tesselata\u003c/em\u003e and their closely related species.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4463026/v1/557d9354180e8426af0edacf.jpg"},{"id":59398982,"identity":"209c76eb-fbdc-4a8f-8b77-756b0e3cfaf9","added_by":"auto","created_at":"2024-07-01 09:44:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":930287,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4463026/v1/45194446-67ba-427f-aaef-fc2669eeb9be.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation and Identification of Endophytic Fungi from the Roots of two epiphytic orchids: Rhynchostylis retusa (L.) Bl. and Vanda tessellata (L.) Bl","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUnder natural or horticultural environments, an archetypal mutualistic association is established among certain heterogeneous group of fungi and orchid roots termed as orchid mycorrhizae (OM). The term mycorrhiza (Fungus Root) was first coined by Professor A. B. Frank in 1885. Systematic studies on OM started more than a century ago and established the fundamental fact that mycorrhizal association is universal in the Orchidaceae, it facilitates nutrients assimilation, seed germination, and act as biological barriers against pathogens (Harris-Valle et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hossain \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). No ̈el Bernard judiciously observed OM and hypothesized that fungus and hosts are nutritionally reliant to each other (Selosse et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Frank hypothesized that orchid mycorrhizae represent a pervasive mutualistic symbiosis where fungus extracts mineral nutrients from surrounding environment and translocate them to their host plants; and in turn the host plants give shelter and nourish the fungi (Yam et al. 2009). Mycorrhizas are classified under 7 groups \u003cem\u003ei.e\u003c/em\u003e., Arbuscular mycorrhiza (AM), Arbutoid mycorrhiza, Ectomycorrhiza (EM), Ericoid mycorrhiza, Monotropid mycorrhiza, Ectendo mycorrhiza and Orchid mycorrhiza (Bellgard et al. 2011). Orchid mycorrhiza is very unique in their structure and functions.\u003c/p\u003e \u003cp\u003eMycorrhizal endophytes colonize in the cortex cells of orchid roots and develop a dense coil like structure by fungal hyphae are known as \"peloton\". Inside the host cells the pelotons are bounded by a membrane of interfacial matrix material. The difference between this membrane and plasma membrane is that this surrounding membrane lacks adenylate cyclase activity. The alignment of cell wall microfibrils and microtubules is changed during infection and may be essential for alteration in the cytoplasm and synthesis of the surrounding membrane of the pelotons. The activity of ascorbic acid oxidase, peroxidase, catalase and polyphenol oxidase increased mycorrhizal infection and help in pelotons digestion in the host cells. Even with their dependency on these mycorrhizal fungi, orchids can control the level of mycorrhizal infection. They regulate fungal growth through the release of a specialized phytotoxin \u0026lsquo;Orchinol\u0026rsquo; which is believed to inhibit fungal growth (Hadley \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Some other literatures suggest that the mature pelotons are digested as a result of the activity of the lytic enzyme \u0026lsquo;orchinol\u0026rsquo; (phytoalexins) produced by the host and release nutrients for host plants. Once the mycorrhizal symbiosis established between orchid roots and fungi, the fungi served as dominant microorganisms and release antagonistic chemical substances, effective in controlling the invasion of other pathogens and enhance growth and survival of seedlings (Hossain et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hossain \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe fungus provides carbohydrates, vitamins and growth stimulus that meet up the nutrient demand of germinating seeds. Fungal exudates stimulate the growth rate as well as the germination percentage \u003cem\u003ein vitro\u003c/em\u003e. Symbiotic seed germination of terrestrial orchids has been used as an alternative to asymbiotic methods with success (Rasmussen \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Johnson et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Jolman et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mycorrhizal fungi provide the basic organic carbon and carbohydrates for seedling growth. Adult individuals of achlorophyllous and photosynthetic orchids obtain part of carbon heterotrophically by mycorrhizal fungi (Bidartondo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Julou et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Selosse et al. 2009; Jacquemyn et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Orchids also receive some organic compounds other than carbon from their fungal partners. \u003cem\u003eGoodyera repens\u003c/em\u003e associated with mycorrhizal fungi acquired 100 times more phosphorus than non-mycorrhizal controls (Alexander et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Phosphorus and Nitrogen (as glycine) transfer from fungus to plant was confirmed through radio labelling experiments (Cameron et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Mycorrhizal fungi may also be a key source of water for orchids. In both the terrestrial \u003cem\u003ePlatanthera integrilabia\u003c/em\u003e and the epiphytic \u003cem\u003eEpidendrum conopseum\u003c/em\u003e water content was higher for mycorrhizal seedlings than uncolonized controls (Yoder et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In adult photosynthetic orchids nitrogen. phosphorus and water continue to flow from the fungal partner but carbon exchange is essentially reversed with photosynthetic providing incentive for continued fungal colonization. Endophytic fungal associations increase host plant fitness to abiotic stresses (Redman et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Bae et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and improve plant adaptability to various environmental conditions (Bonnardeaux et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Swarts et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom the beginning of orchid mycorrhizal research, it was believed that \u003cem\u003eRhizoctonia\u003c/em\u003e is the only fungal partner associated with orchids. Afterward, a number of diverse fungi were isolated and identified from different orchids which commonly termed as \u003cem\u003eRhizoctonia\u003c/em\u003e-like fungi because they exhibit similarity in many characteristics to \u003cem\u003eRhizoctonia\u003c/em\u003e (Bhuiyan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hossain \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recently orchid mycorrhizal fungi are using for biohardening of \u003cem\u003ein vitro\u003c/em\u003e grown plantlets to enhance survival rates to \u003cem\u003eex vitro\u003c/em\u003e environment (Hossain et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, 2022). They also have remarkable role in enhancing both reproductive and vegetative growth of orchid plants; stimulate early flowering, improve flower quality; and reduce severity if disease infection (Hossain \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The aim of the present research program is to isolate and identify OMF from two important indigenous orchids of Bangladesh namely, \u003cem\u003eRhynchostylis retusa\u003c/em\u003e and \u003cem\u003eVenda tesselata\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eFresh mycorrhizal roots of \u003cem\u003eVanda tessellata\u003c/em\u003e and \u003cem\u003eRhynchostylis retusa\u003c/em\u003e were collected in air-tight plastic bags from the naturally grown plants during November-December (winter season) and May-June (rainy season) and used for isolation of fungal endophytes within 24 hrs of collection. The root samples were washed under running tap water to remove dust particles and debrides from root surface and cut into thin transverse sections at different portions of root and observed under microscope by staining with lactophenol cotton blue to confirm the presence of fungal pelotons. The roots showed the occurrence of fungal pelotons were used for isolation of fungal endophytes. The occurrence of fungal endophytes and formation of pelotons in the root cortex cells were calculated by following formula (Hossain \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e% fungal colonized cells in the root cortex\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$=\\frac{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{c}\\text{e}\\text{l}\\text{l}\\text{s} \\text{s}\\text{h}\\text{o}\\text{w}\\text{i}\\text{n}\\text{g} \\text{c}\\text{o}\\text{l}\\text{o}\\text{n}\\text{i}\\text{z}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\text{p}\\text{e}\\text{r} \\text{m}\\text{i}\\text{c}\\text{r}\\text{o}\\text{s}\\text{c}\\text{o}\\text{p}\\text{i}\\text{c} \\text{v}\\text{i}\\text{e}\\text{w} }{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l} \\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{c}\\text{e}\\text{l}\\text{l}\\text{s} \\text{p}\\text{e}\\text{r} \\text{m}\\text{i}\\text{c}\\text{r}\\text{o}\\text{s}\\text{c}\\text{o}\\text{p}\\text{i}\\text{c} \\text{v}\\text{i}\\text{e}\\text{w}}\\times 100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe roots showed the occurrence of fungal pelotons were washed with distilled water and treated by 0.1% HgCl\u003csub\u003e2\u003c/sub\u003e for 6 to 10 mins for surface sterilization. Afterword these were washed with sterile distilled water for 3 times to remove mercuric chloride solution. Final disinfection of the roots was completed by dipping in 70% ethanol for 30 sec to 1 min and washed thoroughly 3 times by double sterile distilled water. The sterilized mycorrhizal roots were then aseptically cut transverse sections approximately 1\u0026ndash;2 mm thickness and cultured in Petri dishes containing potato dextrose agar (PDA) medium. The culture plates were incubated at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026deg;C in an incubator until fungi were grown visibly from root sections onto the PDA surface. The fungi that only grown from the inner portion of the cultured root sections were taken in consideration as possible mycorrhizal fungi. Pure fungal cultures were achieved through transferring a bit of hyphal tips from actively growing fungal colony onto fresh PDA medium. Nature of fungal colony growth, both surface and reverse colours of the colony at young and mature stages were recorded. The diameter of the hyphae and dimensions of spores or monilioid cells were measured by light microscope (Nikon E600, Tokyo, Japan) through mounting the mycelium with lactophenol cotton blue taken on glass slides. Colony growth rates were measured according to Currah et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) by inoculating uniform mycelial bits at the middle of petri plates containing PDA medium and represented by averages based on three replications. Radial increments in colony size were measured at 48 hrs interval over two weeks. For determining nuclei number per vegetative cell, spore or monilioid cell; a small part of the mycelial mat was taken on a glass slide, fixed in 2% formaldehyde for 2 min and cleaned with sterile distilled water for 1 min, followed by staining for 10 min with gold antifade diamidino-2-phenylindole (DAPI, ProLong\u0026reg;, Invitrogen Ltd., Eugene, USA), and destained with sterile distilled water for 2 min. Thereafter, a drop of 50% glycerin was spread over the DAPI stained sample, protected by cover slip and observed under microscope equipped with fluorescence accessory with mercury lamp. Micrographs of different fungal parts were taken by Nikon E600 microscope furnished with photographic accessories.\u003c/p\u003e \u003cp\u003eFor extraction of genomic DNA 1g mycelial mat was taken from two weeks old fungal cultures following the protocol described by Liu et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The ITS1, 5.8S ribosomal RNA gene and ITS2 were amplified using forward and reverse primers ITS1 (5\u0026rsquo; TCCGTAGGTGAACCTGCGG) and ITS4 primers (5\u0026rsquo;GCTGCGTTCATCGATGC) (White et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The PCR master mix was prepared in 50 \u0026micro;l reaction matrix containing 2 \u0026micro;l of genomic DNA, 5 \u0026micro;l of dNTPs mixture, 1 \u0026micro;l of each primer (10 pmol), 5 \u0026micro;l of 10\u0026times; PCR buffer, 0.4 \u0026micro;l of Taq polymerase 1U, and 35.6 \u0026micro;l of MiliQ water. The PCR cycle condition was an initial denaturation at 94\u0026deg;C for 5 min following 35 cycles of denaturation at 94\u0026deg;C for 30 sec, annealing at 55\u0026deg;C for 45 sec and final extension at 72\u0026deg;C for 7 min.\u003c/p\u003e \u003cp\u003eThe PCR amplicons were purified, concentrated and generated the nucleotide sequences of the PCR products by a DNA Analyzer (Thermo Fisher Scientific, McLab, California, USA). A consensus sequence was generated from the forward and reverse sequences by using BioEdit 7.2.6 software (Hall, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The consensus sequence was deposited in the NCBI GenBank (Accession no. OQ073691 and OP752102). The consensus sequence was placed into web based Basic Local Alignment Search Tool (BLAST) of NCBI GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to find out similar species/isolates. The phylogenetic relationships were established based on maximum parsimony tree by using analysis program of the MEGA version 11.0.13 software (Tamura et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this analysis Bootstrap replications number was 1000.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eTwo fungal endophytes, one from each orchid species, were isolated from the mature roots of the epiphytic orchids namely, \u003cem\u003eRhynchostylis retusa\u003c/em\u003e (L.) Bl. and \u003cem\u003eVanda tessellata\u003c/em\u003e (L.) Bl. The fungal endophytes showed intra- and inter-cellular colonization of the cortical cells of naturally grown plants with pelotons formation within the matured roots of the selected orchids. Fungal colonization was detected only in the root portions attached to the substratum in both \u003cem\u003eV. tessellata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and \u003cem\u003eR. retusa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The mycorrhizal colonization in the selected orchids were occupied a considerable proportion of the cortex cells. No pelotons were found in the root tip regions in both cases. The pelotons appeared in the colonized roots as loose coils surrounded by live hyphae in the outer-cortex considered as young pelotons and brownish in the inner cortex which considered as old pelotons. The percentage of fungal colonization differed in different growing seasons. The incidence of root colonization by fungi was significantly higher in rainy season (June-July) than dry season (November-December) in both the orchid species. In case of \u003cem\u003eR. retusa\u003c/em\u003e, the root cortex cells colonization percentage was 95.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99% in rainy season and 76.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22% in winter seasons while in the roots of \u003cem\u003eV. tessellata\u003c/em\u003e showed 88.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55% cell colonization in rainy season and 65.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07% in winter season. The percentage of fungal colonization in orchid roots also varies according to the root types. Bertolini et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) demonstrated that mycorrhization is meticulously extant in orchid roots and it is dependent on the wet and dry seasons. They also checked that rainfall determines higher fungal colonization and the presence of greater number of fresh and undigested live pelotons in the roots. Similar findings were also reported in \u003cem\u003eGastrochilus calceolaris\u003c/em\u003e (Hossain \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and \u003cem\u003eR. retusa\u003c/em\u003e (Bhuiyan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and \u003cem\u003eVenda tesselata\u003c/em\u003e (Shagufta et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The greater intensity of mycorrhizal colonization in rainy season and thoroughgoing occurrence of pelotons in root portions that directly contact with the substrate is reported in epiphytic orchids, \u003cem\u003eEpidendrum stamfordianum\u003c/em\u003e, \u003cem\u003eErycina crista-galli\u003c/em\u003e, \u003cem\u003eStelis quadrifida\u003c/em\u003e (Bertolini et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Reasons behind maximum cell colonization by fungi during summer and rainy seasons for vigorous vegetative growth and flowering of plant as compared to the winter season of slow growth and dry weather. These observations validated the previous reports of higher colonization by well-matched fungi to meet up the additional nutrient demand for vigorous growth and flowering of orchids (Hossain \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Buiyan et al. 2021). Mycorrhizal fungi are sensitive to environmental conditions and seasonal effects influence the structures of mycorrhizal association in the orchid (Han et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). They also noticed that the decrease of fungal population density and change the leading associative fungi across the two different seasons; for example, the common OMF \u003cem\u003eTulasnella\u003c/em\u003e and \u003cem\u003eRussula\u003c/em\u003e significantly reduced their abundance in the dry season. The pelotons in the inner-cortex displayed brownish coloured loose hyphal coils as a result of breakdown of fungal hyphae by the activity of lytic enzymes produced by the host cells and discharge nutrients. Orchids also show sensible defense mechanisms for controlling the level of fungal penetration and colonization by producing phytoalexins or orchinol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eColony growth appearance of fungi, colour of the young and mature colony in both front and reverse view and growth rate were studied. In the case of fungi isolated from \u003cem\u003eR. retusa\u003c/em\u003e, cottony growth of the colony was observed. The young colonies were white colour at the front side (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and off-white colour at the reverse side. The old colonies were greyish on the front side and brown at the back side (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,D). In the case of the fungi isolated from \u003cem\u003eV. tessellata\u003c/em\u003e, cottony appearance of the fungal colony was found. The young colonies were white at the front side (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and greyish white at the back side. The old colonies were slightly brownish at the front side (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and yellowish at the reverse side. The average colony growth rate of the fungi isolated from \u003cem\u003eR. retusa\u003c/em\u003e was 0.26 mm/h while in the fungi isolated from \u003cem\u003eV. tessellata\u003c/em\u003e was 0.30 mm/h.\u003c/p\u003e \u003cp\u003eThe microscopic features of the fungal endophytes \u003cem\u003ei.e\u003c/em\u003e. hyphal structure, hyphal diameter, shape, size and diameter of monilioid cells, branching pattern, colour of the vegetative hyphae of the fungal endophyte isolated from \u003cem\u003eR. retusa\u003c/em\u003e exhibited resemblance with the anamorphic \u003cem\u003eRhizoctonia\u003c/em\u003e-like fungus \u003cem\u003eCeratobasidium\u003c/em\u003e sp. The monilioid cells were elipsoidal and in chain form with slightly thicker wall than the vegetative hyphae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The endophytes showed a slight constriction at each hyphal branching point with a dolipore septum subtending the hyphal branch slightly above the constriction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In case of the fungus isolated from the roots of \u003cem\u003eV. tessellata\u003c/em\u003e, the colour of the hyphae was also hyaline. The hyphae were less than 5\u0026micro;m in diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This isolate had no special hyphal character as like the fungi isolated from \u003cem\u003eR. retusa\u003c/em\u003e. Monilioid cells were absent but huge number of round shaped microspores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), multicellular macrospores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) and round shape chlamydospores were produced. The spore morphology showed resemblance to \u003cem\u003eFusarium\u003c/em\u003e sp.\u003c/p\u003e \u003cp\u003eOther than mycorrhiza forming fungi, there are many endophytic and surface associated fungi were documented from orchid roots. These associated fungi are especially abundant in terrestrial orchid roots. The non-mycorrhizal orchid endophytic fungi are more diverse than \u003cem\u003eRhizoctonia\u003c/em\u003e-like fungi which comprise over 110 genera, for example, \u003cem\u003eFusarium, Trichoderma, Armillaria, Cochliobolus, Sclerotiana\u003c/em\u003e, \u003cem\u003eMerismodes, Hypocrea\u003c/em\u003e, etc. (Ma et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The role of non-mycorrhizal orchid endophytes is comparatively less explored. It is presumed that non-mycorrhizal endophytes play a vital role in growth and development of orchids. Fungal endophytes such as \u003cem\u003eAlternaria\u003c/em\u003e sp., \u003cem\u003eFusarium oxysporum\u003c/em\u003e isolated from terrestrial orchids in Brazil (Vaz et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). From the beginning of orchid mycorrhizal study, only \u003cem\u003eRhizoctonia\u003c/em\u003e-like fungi \u003cem\u003ei.e\u003c/em\u003e. \u003cem\u003eCeratobasidium\u003c/em\u003e, \u003cem\u003eThanatephorus\u003c/em\u003e, \u003cem\u003eTulasnella\u003c/em\u003e, \u003cem\u003eSebacina, Mycena\u003c/em\u003e etc. was considered OMF. Subsequently, some other fungal species such as \u003cem\u003eTrichoderma\u003c/em\u003e spp., \u003cem\u003eFusarium\u003c/em\u003e sp., \u003cem\u003eAspergillus\u003c/em\u003e spp., \u003cem\u003ePiriformospora\u003c/em\u003e sp., \u003cem\u003eSerendipita\u003c/em\u003e sp., \u003cem\u003eGymnopus\u003c/em\u003e sp. have been isolated from orchids as root endophytes (Hossain \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Very recently, \u003cem\u003eFusarium\u003c/em\u003e sp. has been reported as a potential and common endophytic fungus from different orchids including \u003cem\u003eVenda tesselata\u003c/em\u003e (Rasmussen et al. 2001; Behera et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Jiang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fuji et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hern\u0026aacute;ndez-Mart\u0026iacute;nez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tian et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe morphological identity of the above fungal endophytes was further confirmed through molecular techniques by sequencing and analysis of Internal Transcribed Spacer (ITS) sequences of the nuclear ribosomal DNA (nrDNA) of the endophytes. The BLASTn search of ITS region sequences of the fungal endophytes isolated from \u003cem\u003eR. retusa\u003c/em\u003e showed maximum similarity (97%) with \u003cem\u003eCeratobasidium papillatum\u003c/em\u003e (GeneBank Accession No. OQ073691), while the fungal endophytes isolated from \u003cem\u003eV. tesselata\u003c/em\u003e exhibited maximum resemblance (99%) with \u003cem\u003eFusarium ambrosium\u003c/em\u003e (GeneBank Accession No. OP752102). The phylogenetic tree based on ITS region sequences of the fungal endophytes and their closely related species formed two distinct groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The ITS region sequencing is a widely used and powerful molecular technique for precise identification of OMF (Sharon et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hossain \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This region is considered as a universal \u0026lsquo;barcode\u0026rsquo; for fungal identification because it has some special features that make it a strong candidate for barcoding. For example, it is easy to amplify because of its high copy number, comparatively few primer sets are required, it has highly conserved small sub-unit and large sub-unit neighboring regions, and varies dramatically between species but relatively little within species, and far better represented than other loci in fungi in the GenBank (Taylor and McCormick \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zettler and Corey \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hossain \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, molecular characterization validated the morphological information. This study also provides new information about \u003cem\u003eFusarium\u003c/em\u003e as root associated fungi in orchids and the techniques adapted for this fungus will facilitate to isolate and precise identification of other orchids mycorrhizal fungi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMM Hossain made substantial contributions to the conception and design of the study and drafted the manuscript; MA Sayem and MS Miah completed the experiment and contributed to writing the manuscript. All authors approved the draft of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors gratefully acknowledge the \u0026lsquo;Research and Publication Cell\u0026rsquo; of the Chittagong University, Bangladesh for providing financial support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlexander C, Alexander IJ, Hadley G (1984) Phosphate uptake by \u003cem\u003eGoodyera repens\u003c/em\u003e in relation to mycorrhizal infection. New Phytol 97(3):401-411\u003c/li\u003e\n\u003cli\u003eBae H, Kim S, Sicher JRC, Kim MS, Strem MD, Bailey BA (2008) The beneficial endophyte, \u003cem\u003eTrichoderma hamatum\u003c/em\u003e, delays the onset of drought stress in Theobroma cacao. Biological Control 46:24-35\u003c/li\u003e\n\u003cli\u003eBehera D, Tayung K, Mohapatra U\u0026Aacute; (2013) PCR-based identification of endophytes from three orchid species collected from Similipal Biosphere Reserve, India. 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Curr Sci 109(1):72-87\u003c/li\u003e\n\u003cli\u003eRasmussen HN (1995) Terrestrial orchids: from seed to mycotrophic plant, Cambridge University Press\u003c/li\u003e\n\u003cli\u003eRasmussen, HN (2002) Recent developments in the study of orchid mycorrhiza. Plant \u0026amp; Soil 244:149-163\u003c/li\u003e\n\u003cli\u003eRedman RS, Sheehan KB, Stout RG, Rodriguez RJ, Henson JM (2002) Thermotolerance generated by plant/fungal symbiosis. Science 298(5598):1581-1581\u003c/li\u003e\n\u003cli\u003eSelosse MA, Boullard B, Richardson D (2011) No\u0026euml;l Bernard (1874\u0026ndash;1911): Orchids to symbiosis in a dozen years, one century ago. Symbiosis 54(2):61-68\u003c/li\u003e\n\u003cli\u003eSelosse MA, Roy M (2009) Green plants that feed on fungi: facts and questions about mixotrophy. Trends in Plant Sci 14:64-70\u003c/li\u003e\n\u003cli\u003eShagufta S, Arun R, Siddique S, Raghuvanshi A (1993) Seasonal changes in \u003cem\u003eVanda tessellata\u003c/em\u003e mycorrhizae. 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Plant Biotechnol Rep 3:1\u0026ndash;56\u003c/li\u003e\n\u003cli\u003eYoder JA, Zettler LW, Stewart SL (2000) Water requirements of terrestrial and epiphytic orchid seeds and seedlings, and evidence for water uptake by means of mycotrophy. Plant Sci 156:145-150\u003c/li\u003e\n\u003cli\u003eYuan ZL, Chen YC, Yang Y (2009) Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic, medicinal orchid (\u003cem\u003eDendrobium nobile\u003c/em\u003e): Estimation and characterization. World J Microb Biotech 25(2):295-303\u003c/li\u003e\n\u003cli\u003eZettler LW, Corey LL (2018) Orchid mycorrhizal fungi: isolation and identification techniques. In: Orchid propagation: from laboratories to greenhouses\u0026mdash;methods and protocols. Humana Press, New York, NY. pp 27-59\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Orchid mycorrhiza, Epiphytic orchid, ITS sequencing, Fusarium, Ceratobasidium","lastPublishedDoi":"10.21203/rs.3.rs-4463026/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4463026/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTwo fungal endophytes were isolated and identified from two indigenous orchids of Bangladesh namely, \u003cem\u003eRhynchostylis retusa\u003c/em\u003e (L.) Bl. and \u003cem\u003eVanda tessellata\u003c/em\u003e (L.) Bl. Nature of fungal colonization, seasonal variations of colonization in root cortex cells were also studied. The fungal endophytes isolated from two different orchids differed in their cultural morphology and microscopic features such as colony morphology, colour of the colony, presence/absence of monilioid cells or spores and diameter of vegetative hyphae. The microscopic features \u003cem\u003ei.e\u003c/em\u003e., hyphal structure, right-angle branching of hyphae, slight constriction at the branching point, shape and diameter of monilioid cells of the endophytic fungus isolated from \u003cem\u003eR. retusa\u003c/em\u003e showed resemblance with the anamorphic \u003cem\u003eRhizoctonia\u003c/em\u003e-like fungi \u003cem\u003eCeratobasidium\u003c/em\u003e sp. On the other hand, the fungal endophytes isolated from the roots of \u003cem\u003eV. tessellata\u003c/em\u003e produced huge number of spores. The cultural characteristics and spore morphology of this fungi corroborated resemblance with pathogenic fungi, \u003cem\u003eFusarium\u003c/em\u003e sp. The identity of the fungi was further reconfirmed through sequencing of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA (nrDNA). The BLASTn search of ITS region sequences of the endophytic fungi isolated from \u003cem\u003eR. retusa\u003c/em\u003e exhibited maximum similarity (97%) with an orchid mycorrhizal fungi (OMF), \u003cem\u003eCeratobasidium papillatum\u003c/em\u003e (GeneBank Accession No. OQ073691), while the fungal endophyte isolated from \u003cem\u003eV. tessellata\u003c/em\u003e showed maximum similarity (99%) with \u003cem\u003eFusarium ambrosium\u003c/em\u003e (GeneBank Accession No. OP752102). The phylogenetic tree constructed using ITS region sequences of the isolated fungal endophytes and their closely linked species from genebank data developed two distinct groups. The conventional and molecular approaches applied for identification of these OMF can be followed for easy and accurate identification of other OMF.\u003c/p\u003e","manuscriptTitle":"Isolation and Identification of Endophytic Fungi from the Roots of two epiphytic orchids: Rhynchostylis retusa (L.) Bl. and Vanda tessellata (L.) Bl","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-05 06:41:21","doi":"10.21203/rs.3.rs-4463026/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":"d70471db-901a-4eeb-9c1e-2f422caa1583","owner":[],"postedDate":"June 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-01T09:36:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-05 06:41:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4463026","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4463026","identity":"rs-4463026","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}