Orchid Hosts Research Articles

Exploration and Characterization of Host Trees and Orchid Epiphyte Zones in Lore Lindu National Park, Central Sulawesi

Host trees are vital for the survival of epiphytic orchids, providing necessary light and air circulation for optimal growth. Large trees with tall canopies contribute to the broader distribution of epiphytic orchids in forest areas. This research aimed to identify the characteristics of orchid host tree species in Lore Lindu National Park, considering both elevation zones and orchid growth zones. The research was conducted at various locations within the park, employing the field exploration survey method (cruise method). This method involved comprehensive exploration at each research site and establishing observation paths along pioneer lines, mainly where orchid host trees were found. The findings indicate the presence of 20 types of orchid host trees and 35 species of epiphytic orchids. The basal branching zone (Zone 3) was the most populated by epiphytic orchids on host trees, and the lowland forest was the elevation zone where most orchid host trees were found in Lore Lindu National Park, Central Sulawesi. Keywords: host trees, Lore Lindu National Park, orchids, zone distribution

Diversity of Mycorrhizal Fungi in Temperate Orchid Species: Comparison of Culture-Dependent and Culture-Independent Methods.

Many orchid species are endangered due to anthropogenic pressures such as habitat destruction and overharvesting, meanwhile, all orchids rely on orchid mycorrhizal fungi (OMF) for seed germination and seedling growth. Therefore, a better understanding of this intimate association is crucial for orchid conservation. Isolation and identification of OMF remain challenging as many fungi are unculturable. In our study, we tested the efficiency of both culture-dependent and culture-independent methods to describe OMF diversity in multiple temperate orchids and assessed any phylogenetic patterns in cultivability. The culture-dependent method involved the cultivation and identification of single pelotons (intracellular hyphal coils), while the culture-independent method used next-generation sequencing (NGS) to identify root-associated fungal communities. We found that most orchid species were associated with multiple fungi, and the orchid host had a greater impact than locality on the variability in fungal communities. The culture-independent method revealed greater fungal diversity than the culture-dependent one, but despite the lower detection, the isolated fungal strains were the most abundant OMF in adult roots. Additionally, the abundance of NGS reads of cultured OTUs was correlated with the extent of mycorrhizal root colonization in orchid plants. Finally, this limited-scale study tentatively suggests that the cultivability character of OMF may be randomly distributed along the phylogenetic trees of the rhizoctonian families.

Destructive Phytophthora on orchids: current knowledge and future perspectives.

Anton de Bary first coined the genus, Phytophthora, which means "plant destroyer", viewing its devastating nature on potatoes. Globally plants have faced enormous threat from Phytophthora since its occurrence. In fact, a century ago, Phytophthorapalmivora was first reported on Dendrobium maccarthiae in Sri Lanka. Since then, members of beautiful flowering crops of the family Orchidaceae facing the destructive threat of Phytophthora. Several Phytophthora species have been recorded to infect orchids with economic loss worldwide. To date, orchids are attacked by 12 species of Phytophthora. Five Phytophthora species (P. palmivora, P. nicotianae, P. cactorum, P. multivesiculata, P. meadii) are the major pathogenic Oomycetous Chromista" rather than true fungi frequently occurred on Orchidaceae. Phytophthora palmivora (having ~32 orchid host genera in 15 countries), Phytophthora nicotianae (having ~15 orchid host genera in 16 countries), Phytophthora cactorum (having ~43 orchid host genera in 6 countries), Phytophthora multivesiculata (having 2 orchid host genera in 5 countries) and Phytophthora capsici (having 2 orchid host genera in all Vanilla growing countries) are potential destroyers of Orchidaceae. Most of them are water loving Oomycetes cause disease in moist environments (> 80% RH) at 16-28°C. In artificially constructed orchidaria, anthropogenic factors are mostly contributed to the dissemination Phytophthora diseases in addition to many other factors. Water management, clean cultivation, and agro-chemicals are the major options for effective management of orchid Phytophthora, as the eco-friendly management options like development of resistant hybrids/cultivars, biological disease management, transgenic approaches, RNAi technology remained in the infant stage. In this review, we intended to highlight the insight of Phytophthora diseases associated with the orchid disease with reference to the historical aspect of the diseases, symptoms and signs, the pathogens, taxonomy, geographic distribution, host range within the Orchidaceae, pathogen identification, molecular diagnostics, mating types and races, management options and strategies and future perspectives.

KARAKTERISTIK POHON INANG DAN KEANEKARAGAMAN ANGGREK (Orchidaceae) EPIFIT DI HUTAN LINDUNG LUNUNIANAM, DESA FATU’ULAN, KECAMATAN KI’E, KABUPATEN TIMOR TENGAH SELATAN, PROVINSI NUSA TENGGARA TIMUR

This study was conducted to determine the characteristics of orchid host trees, the diversity of epiphytic orchids (Orchidaceae), and the preservation of host trees and epiphytic orchids in the Lununianam protected forest area. This research was conducted in Lununianam Protection Forest, Fatu'ulan Village, Ki'e District, South Central Timor Regency, East Nusa Tenggara Province. Which is carried out for 1 month, from November to December 2022. This study used purposive sampling methods, surveys, and cruising paths with data analysis using qualitative descriptive and quantitative descriptive analysis. Host tree data was processed using vegetation analysis formulas and orchid data was processed using the Shannon wiener (H') diversity index formula.
 The results showed that there were 5 species of host trees found in the study site, namely forest guava (Syzygium pycnanthum), Matani (Pterocarpus indicus), Tahi (Celtis wightii planch), Kamala (Mallotus philippensis), Jenitri (Elaeocarpus ganitrid). Host trees found in Lununianam Protection forest have the characteristics of large hard and rough bark, have a height range of 12-30 m, and bark overgrown with moss, The results of vegetation analysis of host tree species with the highest Important Value Index namely Syzygium Pycnanthum and Elaeocarpus ganitrus have the lowest Important Value Index. Orchids found in the sampling plot in the Lununianam Protection Forest contained 8 species of epiphytic orchids, namely Dendrobium Sp, Vanda insignis, Eria multiflora, Flickingeria grandiflora, Trichoglottis bipenicillata, Pholidota carnea, Appendicula reflexa Blume, and Ceologyne speciosa. The calculation of Shannon Wiener's diversity index of 1.57 (H') is classified as medium (1<H'<3). The most common type of orchid found is the type Eria multifora with a total of 168 individuals and the type that is less commonly found is Appendicula reflexa Blume with 4 individuals.

Natural Orchid Diversity and Its Conservation Strategies in Wonosobo Aqua Biodiversity Park

This study aims to determine the diversity of natural orchids and to design a strategy for preserving natural orchids in the KEHATI AQUA Park, Wonosobo. The data sought in this study were the type, number, diversity of orchids, and questionnaire respondent data. The results showed that there were three types of natural orchids in KEHATI AQUA Wonosobo Park, namely Dendrobium crumenatum, Eria retusa, and Liparis sp. Overall, the total number of orchids found was 487 individuals. The three types of orchids found were epiphytic orchids found on 6 types of host trees. Based on the questionnaire respondents' data which was carried out by a SWOT analysis, there were several strategies designed, namely optimizing the land for orchid conservation, utilizing existing facilities in cultivation ( green house ), increasing the types of orchids to be cultivated and caring for and maintaining existing vegetation for orchid host trees, making policies aimed at preserving orchids and establishing cooperation with the orchid conservation community. The conclusion of this study is that there are 3 types of natural orchids in KEHATI AQUA Wonosobo Park, namely Dendrobium crumenatum with an INP value of 1.04, Eria Retusa with an INP value of 0.87, and Liparis sp with an INP value of 0.09, a diversity index value of 0.72, an evenness index value of 0.66 and the species richness index value of 0.32 and the alternative strategy used in the conservation of natural orchids in the KEHATI AQUA Park Wonosobo is the SO strategy or a strategy that is made to take advantage of all strengths to seize and take advantage of opportunities as much as possible.

Characteristics of Host Trees as Habitat for Ephiphytic Orchids in the Cibereum waterfall Path, Mount Gede Pangrango, West Java

An epiphytic orchid is one type of orchid that has the characteristics of habitat attached to the host tree. Mount Gede Pangrango, especially in the Cibereum waterfall, Selabintana, is a tropical forest area with many large host trees with a lush canopy, causing the epiphytic orchid species to form be abundant in the area. This study aims to determine the type and characteristics of the host tree used as a habitat for epiphytic orchids in the Cibereum waterfall path. The research method is to make ten plots on the right and left sides of the path, with each plot measuring 500x5 m and the distance between plots is 100 m. The results showed that on the Cibereum waterfall path, 33 species of host trees were found, consisting of 20 families. At the same time, the types of orchids found were 31 species from 12 genera. Almost all of the orchid hosts are trees, except for Brassaiopsis glomerulata are shrubs and Cyathea sp are poles. The characteristics of host trees as habitat for epiphytic orchids are: 4-51 m tall, trunk diameter 7-89 cm, canopy width 0-20 m, bark thickness 0-1.2 cm, bark texture rough to medium, generally colored brown, has no sap except for Ficus variegata, the surface of the trunk contains moss, and the epiphytic orchid growth zone on the host tree is mostly found in zones III and IV.

The Dark Side of Orchid Symbiosis: Can Tulasnella calospora Decompose Host Tissues?

Photosynthetic orchids associate with mycorrhizal fungi that can be mostly ascribed to the “rhizoctonia” species complex. Rhizoctonias’ phylogenetic diversity covers a variety of ecological/nutritional strategies that include, beside the symbiosis establishment with host plants, endophytic and pathogenic associations with non-orchid plants or saprotrophic soil colonization. In addition, orchid mycorrhizal fungi (OMF) that establish a symbiotic relationship with an orchid host can later proliferate in browning and rotting orchid tissues. Environmental triggers and molecular mechanisms governing the switch leading to either a saprotrophic or a mycorrhizal behavior in OMF remain unclear. As the sequenced OMF genomes feature a wide range of genes putatively involved in the degradation of plant cell wall (PCW) components, we tested if these transitions may be correlated with a change in the expression of some PCW degrading enzymes. Regulation of several genes encoding PCW degrading enzymes was evaluated during saprotrophic growth of the OMF Tulasnella calospora on different substrates and under successful and unsuccessful mycorrhizal symbioses. Fungal gene expression in planta was investigated in two orchid species, the terrestrial Mediterranean Serapias vomeracea and the epiphytic tropical Cattleya purpurata. Although we only tested a subset of the CAZyme genes identified in the T. calospora genome, and we cannot exclude therefore a role for different CAZyme families or members inside a family, the results showed that the degradative potential of T. calospora is finely regulated during saprotrophic growth and in symbiosis, often with a different regulation in the two orchid species. These data pose novel questions about the role of fungal PCW degrading enzymes in the development of unsuccessful and successful interactions.

Diversity of mycorrhizal Tulasnella associated with epiphytic and rupicolous orchids from the Brazilian Atlantic Forest, including four new species

The genus Tulasnella often forms mycorrhizas with orchids and has worldwide distribution. Species of this genus are associated with a wide range of orchids, including endangered hosts. Initially, species identification relied mostly on morphological features and few cultures were preserved for later phylogenetic comparisons. In this study, a total of 50 Tulasnella isolates were collected from their natural sites in Minas Gerais, Brazil, cultured, and subjected to a phylogenetic analysis based on alignments of sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA. Our results, based on phylogeny, integrated with nucleotide divergence and morphology, revealed the diversity of isolated Tulasnella species, which included four new species, namely, Tulasnella brigadeiroensis, Tulasnella hadrolaeliae, Tulasnella orchidis and Tulasnella zygopetali. The conservation of these species is important due to their association with endangered orchid hosts and endemic features in the Brazilian Atlantic Forest.

Inventarisasi Jenis Pohon Inang Anggrek Epifit di Desa Tompobulu Resort Balocci Taman Nasional Bantimurung Bulusaraung Kabupaten Pangkep Provinsi Sulawesi Selatan

Abstract. Balocci Resort Banntimurung Bulusaraung National Park is a location that is overgrown with many natural orchids. This research is a descriptive study that aims to determine the type of epiphytic orchid host tree and the distribution of host trees at the Balocci Resort in Bantimurung Bulusearaung National Park. The method used is a survey method. The stages of the research are termining location and observations, collecting data and indetifying spesies. Based on the result; Alstonia scholaris, Sauarauia malayana, Mangifera sp, Tabernaemontana sphaerocarpa, Arenga pinnata, Syzygium sp, Elaecarpus angustifolius, Bischofia javanica, Maesea sp, Erythrina subumbrans, Lithocarpus celebicus, Cryptocarya sp, Elmerillia ovalis, Memecylon paniculatum, Ficus benjamina L, Ficus variegate, Ficus fistulosa, Knema laurina, Litsea sp, Uncaria longiflora, Coffea sp, Melicope sp, Planchonella firma, Palaquium lobbianum, and Laportea stimulant which belongs to 19 families. The tree that is overgrows with orchids is Alstonia scholaris, Sauarauia malayana and Ficus benjamina L. the host tree which is used as a place for growth has uneven surface characteristics of the tree, overgrown with nails and generally mossy. Host tree species that have a wide spread on mount Bulusaraung is Sauarauia malayana which is at an altitude of 700-1300 m above sea level. Keywords: host tree, epiphytic, resort balocci, bantimurung bulusaraung national park area

Infestation of Phalaenopsis aphrodite Rchb.f. (Asparagales: Orchidaceae) flower buds by Japanagromyza tokunagai (Sasakawa) (Diptera: Agromyzidae) in a greenhouse on Shikoku Island, Japan

Abstract To protect endangered and commercially important orchid species, it is important to monitor and accurately identify orchid pests. Japanagromyza tokunagai (Sasakawa) larvae are known to infest the flower buds, stems, and ovaries of various Japanese orchid species. Given that J. tokunagai can infest diverse orchid hosts, J. tokunagai has the potential to be an economically important orchid pest in greenhouses. However, damage by J. tokunagai has previously only been reported in outdoor habitats. Here we revealed that the dipteran larvae infesting the flower buds of Phalaenopsis aphrodite Rchb.f. (Orchidaceae) cultivated in a greenhouse are J. tokunagai based on DNA barcoding, using COI sequences. Infested flower buds remained closed and eventually dropped to the ground, making the P. aphrodite plants commercially worthless. Phalaenopsis aphrodite is one of the most economically important ornamental orchids. Therefore, J. tokunagai is a potential threat to orchid production and we recommend taking precautions to prevent its spread.

Infestation of the orchid Cephalanthera spp. by Parallelomma vittatum (Meigen, 1826) (Diptera: Scathophagidae) in Japan

AbstractIt is important to understand the interactions between orchids and their herbivores, especially considering the rarity of most orchids and the potential negative effects of pests on orchid reproduction. Here, we report the infestation of leaves of Cephalanthera spp. orchids by Parallelomma vittatum (Scathophagidae) in Japan. The present study represents the first report of P. vittatum on orchid hosts in Asia.

A commensal network of epiphytic orchids and host trees in an Atlantic Forest remnant: A case study revealing the important role of large trees in the network structure

AbstractApplication of metrics derived from network theory could elucidate the structural organization of orchid assemblages, and help identify the host tree species on which they depend, as well as predicting the impacts of removing host tree species. In this study, we used nestedness, modularity, connectance and robustness, to identify the factors that predict the structure of a quantitative orchid–host tree network in a remnant of Atlantic Forest in Brazil. The network exhibited low nestedness (NODF = 14.07; P = 0.03; WNODF = 5.3; P = 0.02) and no modularity. It was highly robust to the random elimination of host tree species, but showed low robustness when host trees with more interactions started to be eliminated. The nested pattern found was attributed to a combination of two main host tree traits, height and diameter (given by DBH measure). Thus, our analyses reflect the importance of tall and large host tree species, which seem to play an important role in the network structure, providing a substrate for orchid species with different habitat associations and representing a decisive factor in both nested and robust patterns found.

Epiphytic Orchid Diversity in UB Forest and Opportunities for Orchid Tourism Road Development

The aims of the research were to identify epiphytic orchid diversity and host tree species for the development of orchid tourism trails as a potential tourist attraction in UB Forest. A Floristic survey and questionnaires were conducted in this study. Nine epiphytic orchids were found in UB Forest, namely Liparis viridiflora, Eria hyacinthoides, Aerides sp., Coelogyne sp., Dendrobium lineairifolium , Agrostophyllum sp., Sarcanthus sp., Flickingeria sp. and Eria monostachya . These epiphytic orchids used Dadap ( Erythrina sp.), Jalina ( Ficus sp.), Kukrup ( Engelhardtia spicata ) and Cempaka wangi ( Michelia champaca ) as host species, from Gate 1 to Gunung Mujur Point in UB Forest. Analysis showed that improvement including preserving and improving the population of host tree species, establishing interpretation media for educational purposes, and improving the attractiveness of tourist attractions and facilities, could provide an opportunity to develop an eco-tourism route. Keywords: Ecotourism, Environmental education, Orchids

Seeking the needle in the haystack: Undetectability of mycorrhizal fungi outside of the plant rhizosphere associated with an endangered Australian orchid

Abstract Co-occurrence and abundance of suitable mycorrhizal fungi are expected to be important drivers for orchid seedling establishment and development, as well as mature plant distribution. However, limited information is available on the occurrence and spatial patterns of orchid mycorrhizal fungi in soil independent of the orchid host. In this study, we investigated the in situ distribution of Tulasnella spp. associated with the critically endangered Australian orchid Diuris fragrantissima. We tested and implemented a meta-barcoding approach (fungal ITS1 region) using three soil sources: orchid rhizosphere, orchid-associated bulk soil and bulk soil from the orchid native site. The quality-filtered data set revealed that the occurrence of Tulasnella spp. in situ is restricted to the orchid rhizosphere, suggesting that a limited number of potential recruitment micro-sites with suitable mycorrhizal taxa exists in the D. fragrantissima natural habitat. The meta-barcoding approach also revealed a distinctive fungal community associated with the orchid rhizosphere. Overall, Next Generation Sequencing technology has proven to be a suitable method for large-scale screening of mycorrhizal fungi in orchid-associated soil.

New species of Tulasnella associated with terrestrial orchids in Australia

Recent studies using sequence data from eight sequence loci and coalescent-based species delimitation methods have revealed several species-level lineages of Tulasnella associated with the orchid genera Arthrochilus, Caleana, Chiloglottis, and Drakaea in Australia. Here we formally describe three of those species, Tulasnella prima, T. secunda, and T. warcupii spp. nov, as well as an additional Tulasnella species associated with Chiloglottis growing in Sphagnum, T sphagneti sp. nov. Species were identified by phylogenetic analyses of the ITS with up to 1.3 % sequence divergence within taxa and a minimum of 7.6 % intraspecific divergence. These new Tulasnella (Tulasnellaceae, Cantharellales) species are currently only known from orchid hosts, with each fungal species showing a strong relationship with an orchid genus. In this study, T. prima and T. sphagneti associate with Chiloglottis, while T. secunda associates with Drakaea and Caleana, and T. warcupii associates with Arthrochilus oreophilus.

Fungal and plant gene expression in the Tulasnella calospora-Serapias vomeracea symbiosis provides clues about nitrogen pathways in orchid mycorrhizas.

Orchids are highly dependent on their mycorrhizal fungal partners for nutrient supply, especially during early developmental stages. In addition to organic carbon, nitrogen (N) is probably a major nutrient transferred to the plant because orchid tissues are highly N-enriched. We know almost nothing about the N form preferentially transferred to the plant or about the key molecular determinants required for N uptake and transfer. We identified, in the genome of the orchid mycorrhizal fungus Tulasnella calospora, two functional ammonium transporters and several amino acid transporters but found no evidence of a nitrate assimilation system, in agreement with the N preference of the free-living mycelium grown on different N sources. Differential expression in symbiosis of a repertoire of fungal and plant genes involved in the transport and metabolism of N compounds suggested that organic N may be the main form transferred to the orchid host and that ammonium is taken up by the intracellular fungus from the apoplatic symbiotic interface. This is the first study addressing the genetic determinants of N uptake and transport in orchid mycorrhizas, and provides a model for nutrient exchanges at the symbiotic interface, which may guide future experiments.

Differences in carbon source utilisation by orchid mycorrhizal fungi from common and endangered species of Caladenia (Orchidaceae).

Terrestrial orchids depend on orchid mycorrhizal fungi (OMF) as symbionts for their survival, growth and nutrition. The ability of OMF from endangered orchid species to compete for available resources with OMF from common species may affect the distribution, abundance and therefore conservation status of their orchid hosts. Eight symbiotically effective OMF from endangered and more common Caladenia species were tested for their ability to utilise complex insoluble and simple soluble carbon sources produced during litter degradation by growth with different carbon sources in liquid medium to measure the degree of OMF variation with host conservation status or taxonomy. On simple carbon sources, fungal growth was assessed by biomass. On insoluble substrates, ergosterol content was assessed using ultra-performance liquid chromatography (UPLC). The OMF grew on all natural materials and complex carbon sources, but produced the greatest biomass on xylan and starch and the least on bark and chitin. On simple carbon sources, the greatest OMF biomass was measured on most hexoses and disaccharides and the least on galactose and arabinose. Only some OMF used sucrose, the most common sugar in green plants, with possible implications for symbiosis. OMF from common orchids produced more ergosterol and biomass than those from endangered orchids in the Dilatata and Reticulata groups but not in the Patersonii and Finger orchids. This suggests that differences in carbon source utilisation may contribute to differences in the distribution of some orchids, if these differences are retained on site.

Relationship between soil nutrients and mycorrhizal associations of two Bipinnula species (Orchidaceae) from central Chile.

Mycorrhizal associations are influenced by abiotic and biotic factors, including climate, soil conditions and the identity of host plants. However, the effect of environmental conditions on orchid mycorrhizal associations remains poorly understood. The present study examined how differences in soil nutrient availability are related to the diversity and composition of mycorrhizal fungi associated with two terrestrial orchid species from central Chile. For 12 populations of Bipinnula fimbriata and B. plumosa, OTU (operational taxonomic unit) richness, phylogenetic diversity and community composition of mycorrhizal fungi in root samples were estimated using internal transcribed spacer (ITS) sequences. Then, these mycorrhizal diversity variables were related to soil nutrients and host species using generalized linear models and non-metric multidimensional scaling. Variation in OTU composition of mycorrhizal fungi among sites was explained mainly by orchid host species. Fungi in Tulasnellaceae and Ceratobasidiaceae were isolated from both orchid species, but the former were more frequent in B. fimbriata and the latter in B. plumosa. Soil nutrients and orchid host species had significant effects on OTU richness and phylogenetic diversity. Mycorrhizal diversity decreased in habitats with higher N in both species and increased with P availability only in B. fimbriata The results suggest that soil nutrient availability modulates orchid mycorrhizal associations and provide support for the hypothesis that specialization is favoured by higher soil nutrient availability. Inter-specific differences in mycorrhizal composition can arise due to a geographical pattern of distribution of orchid mycorrhizal fungi, host preferences for fungal partners or differential performance of mycorrhizal fungi under different nutrient availabilities. Further experiments are needed to evaluate these hypotheses.

Characterization of the first two viruses described from wild populations of hammer orchids (Drakaea spp.) in Australia

Sequences representing the genomes of two distinct virus isolates infecting wild plants of two members of the genus Drakaea (hammer orchids) in Western Australia are described. The virus isolated from Drakaea livida has a bipartite genome of 4490 nt (RNA1) and 2905 nt (RNA2) that shares closest sequence and structural similarity to members of the genus Pecluvirus, family Virgaviridae, described from legumes in the Indian subcontinent and West Africa. However, it differs from pecluviruses by lacking a P39 protein on RNA2 and having a cysteine‐rich protein gene located 3′ of the triple gene block protein genes. It is the first peclu‐like virus to be described from Australia. The name Drakaea virus A is proposed (DVA; proposed member of the family Virgaviridae, genus unassigned). The second virus isolate was identified from Drakaea elastica, a species classed as endangered under conservation legislation. The genome sequence of this virus shares closest identity with isolates of Donkey orchid symptomless virus (DOSV; proposed member of the order Tymovirales, family and genus unassigned), a species described previously from wild Caladenia and Diuris orchids in the same region. These viruses are the first to be isolated from wild Drakaea populations and are proposed to have an ancient association with their orchid hosts.

Development of phylogenetic markers for Sebacina (Sebacinaceae) mycorrhizal fungi associated with Australian orchids.

• Premise of the study: To investigate fungal species identity and diversity in mycorrhizal fungi of order Sebacinales, we developed phylogenetic markers. These new markers will enable future studies investigating species delineation and phylogenetic relationships of the fungal symbionts and facilitate investigations into evolutionary interactions among Sebacina species and their orchid hosts.• Methods and Results: We generated partial genome sequences for a Sebacina symbiont originating from Caladenia huegelii with 454 genome sequencing and from three symbionts from Eriochilus dilatatus and one from E. pulchellus using Illumina sequencing. Six nuclear and two mitochondrial loci showed high variability (10–31% parsimony informative sites) for Sebacinales mycorrhizal fungi across four genera of Australian orchids (Caladenia, Eriochilus, Elythranthera, and Glossodia).• Conclusions: We obtained highly informative DNA markers that will allow investigation of mycorrhizal diversity of Sebacinaceae fungi associated with terrestrial orchids in Australia and worldwide.