Extracellular Enzyme Activities and Carbon/Nitrogen Utilization in Mycorrhizal Fungi Isolated From Epiphytic and Terrestrial Orchids

Fungi employ extracellular enzymes to initiate the degradation of organic macromolecules into smaller units and to acquire the nutrients for their growth. As such, these enzymes represent important functional components in terrestrial ecosystems. While it is well-known that the regulation and efficiency of extracellular enzymes to degrade organic macromolecules and nutrient-acquisition patterns strongly differ between major fungal groups, less is known about variation in enzymatic activity and carbon/nitrogen preference in mycorrhizal fungi. In this research, we investigated variation in extracellular enzyme activities and carbon/nitrogen preferences in orchid mycorrhizal fungi (OMF). Previous research has shown that the mycorrhizal fungi associating with terrestrial orchids often differ from those associating with epiphytic orchids, but whether extracellular enzyme activities and carbon/nitrogen preference differ between growth forms remains largely unknown. To fill this gap, we compared the activities of five extracellular enzymes [cellulase, xylanase, lignin peroxidase, laccase, and superoxide dismutase (SOD)] between fungi isolated from epiphytic and terrestrial orchids. In total, 24 fungal strains belonging to Tulasnellaceae were investigated. Cellulase and xylanase activities were significantly higher in fungi isolated from terrestrial orchids (0.050 ± 0.006 U/ml and 0.531 ± 0.071 U/ml, respectively) than those from epiphytic orchids (0.043 ± 0.003 U/ml and 0.295 ± 0.067 U/ml, respectively), while SOD activity was significantly higher in OMF from epiphytic orchids (5.663 ± 0.164 U/ml) than those from terrestrial orchids (3.780 ± 0.180 U/ml). Carboxymethyl cellulose was more efficiently used by fungi from terrestrial orchids, while starch and arginine were more suitable for fungi from epiphytic orchids. Overall, the results of this study show that extracellular enzyme activities and to a lesser extent carbon/nitrogen preferences differ between fungi isolated from terrestrial and epiphytic orchids and may indicate functional differentiation and ecological adaptation of OMF to local growth conditions.

Influence of Culture Conditions on In Vitro Asymbiotic Germination of Anacamptis longicornu and Ophrys panormitana (Orchidaceae)

This study is the first approach to in vitro asymbiotic germination of two species of Sicilian threatened terrestrial orchids, Anacamptis longicornu and Ophrys panormitana. Seeds were collected in the wild and cultured in two different media—Orchimax medium (OM) and Murashige and Skoog (MS)—and exposed to different photoperiods and temperatures to evaluate the best conditions for the specific stages of development. The germination of A. longicornu was very high on OM (95.5%) and lower on MS medium (21.4%), whereas O. panormitana germinated only on OM medium, with significantly lower percentages (12.0%), compared with A. longicornu. This difference is caused by variation in quality and quantity of nutrients used, primarily by nitrogen source. The results show that temperature and photoperiod widely affect seed germination and development. Although further investigations on asymbiotic and symbiotic germination are needed for the improvement of conservation of Mediterranean terrestrial orchids, our results contribute to the conservation of this group of plants.

The effects of endophytic fungi of NZ terrestrial orchids: developing methods for conservation

<p>Nearly 40% of New Zealand (NZ) orchid species are of conservation concern, some critically endangered, largely due to habitat loss. In NZ, there are currently no propagation programs for terrestrial orchids all of which rely on symbiotic fungi to provide the nutrients required for germination, and little is known about the specific fungal species that might make this possible. To develop an understanding of the fungal interactions affecting recruitment in the field, a survey of endophytic fungal diversity from the roots of Chiloglottis valida, Microtis unifolia, Pterostylis banksii, Spiranthes novae-zelandiae and Thelymitra longifolia was carried out. The identification of fungi was assisted by obtaining sequences of the ITS rDNA gene marker. Seeds of M. unifolia, P. banksii, S. novae-zelandiae and T. longifolia were inoculated with cultured endophytes that were recovered from the roots of conspecific orchids, and their effect on seed germination evaluated. Seed viability using fluorescein diacetate was assayed on all species prior to all experiments and showed moderate to high viability scores for all species. Recovered endophytes belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The effect of the different endophytes on seed germination was variable, with five inoculants exhibiting a positive response. Three inoculants had a consistent negative effect on seed germination. The distribution of orchid symbiotic mycorrhizae in situ was investigated at Otari-Wilton’s Bush, Wellington, NZ. Mesh seed packets containing seed of M. unifolia and T. longifolia were interred for 150 days, along transects (≤ 1 metre) that originated at adult orchids at three sites, and an additional site with no adult orchids was used as a control. No small-scale patterns were detected; however, germination rates were higher at undisturbed sites. Seed viability was considerably reduced to <2% after five months under the soil suggesting M. unifolia and T. longifolia seeds do not persist in the seed bank beyond one growing season. Sequences of ITS rDNA indicate Tulasnella calospora assists in the germination of M. unifolia at this site. Similarly, Tulasnella calospora promoted germination of the Nationally Vulnerable wetland species S. novae-zelandiae. Pelotons were isolated from the roots of S. novae-zelandiae plants from a wild population from the lower north island and cultured in Petri dishes. Germination of this orchid began after 30 days from inoculation when the pelotons are already observed inside the embryo. Chlorophyllus tissue was observed after c. 80 days of inoculation. The phylogenetic relationship of Asian-Pacific Spiranthes species with New Zealand Spiranthes was also investigated using nuclear (ITS) and chloroplast (trnL-trnF) DNA sequences. Phylogenetic analyses supported the recognition of Spiranthes novae-zelandiae ‘Motutangi’ as a distinct taxonomic unit. It was also found that the Asian-Pacific Spiranthes species are in need of taxonomic revision. Methods used and developed in this thesis study could be used to identify potential orchid symbionts and pathogens, assess suitable potential relocation sites, and propagation of NZ orchids using symbiotic fungi for restoration and conservation purposes.</p>

The effects of endophytic fungi of NZ terrestrial orchids: developing methods for conservation

<p>Nearly 40% of New Zealand (NZ) orchid species are of conservation concern, some critically endangered, largely due to habitat loss. In NZ, there are currently no propagation programs for terrestrial orchids all of which rely on symbiotic fungi to provide the nutrients required for germination, and little is known about the specific fungal species that might make this possible. To develop an understanding of the fungal interactions affecting recruitment in the field, a survey of endophytic fungal diversity from the roots of Chiloglottis valida, Microtis unifolia, Pterostylis banksii, Spiranthes novae-zelandiae and Thelymitra longifolia was carried out. The identification of fungi was assisted by obtaining sequences of the ITS rDNA gene marker. Seeds of M. unifolia, P. banksii, S. novae-zelandiae and T. longifolia were inoculated with cultured endophytes that were recovered from the roots of conspecific orchids, and their effect on seed germination evaluated. Seed viability using fluorescein diacetate was assayed on all species prior to all experiments and showed moderate to high viability scores for all species. Recovered endophytes belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The effect of the different endophytes on seed germination was variable, with five inoculants exhibiting a positive response. Three inoculants had a consistent negative effect on seed germination. The distribution of orchid symbiotic mycorrhizae in situ was investigated at Otari-Wilton’s Bush, Wellington, NZ. Mesh seed packets containing seed of M. unifolia and T. longifolia were interred for 150 days, along transects (≤ 1 metre) that originated at adult orchids at three sites, and an additional site with no adult orchids was used as a control. No small-scale patterns were detected; however, germination rates were higher at undisturbed sites. Seed viability was considerably reduced to <2% after five months under the soil suggesting M. unifolia and T. longifolia seeds do not persist in the seed bank beyond one growing season. Sequences of ITS rDNA indicate Tulasnella calospora assists in the germination of M. unifolia at this site. Similarly, Tulasnella calospora promoted germination of the Nationally Vulnerable wetland species S. novae-zelandiae. Pelotons were isolated from the roots of S. novae-zelandiae plants from a wild population from the lower north island and cultured in Petri dishes. Germination of this orchid began after 30 days from inoculation when the pelotons are already observed inside the embryo. Chlorophyllus tissue was observed after c. 80 days of inoculation. The phylogenetic relationship of Asian-Pacific Spiranthes species with New Zealand Spiranthes was also investigated using nuclear (ITS) and chloroplast (trnL-trnF) DNA sequences. Phylogenetic analyses supported the recognition of Spiranthes novae-zelandiae ‘Motutangi’ as a distinct taxonomic unit. It was also found that the Asian-Pacific Spiranthes species are in need of taxonomic revision. Methods used and developed in this thesis study could be used to identify potential orchid symbionts and pathogens, assess suitable potential relocation sites, and propagation of NZ orchids using symbiotic fungi for restoration and conservation purposes.</p>

Inventarisasi Jenis-Jenis Anggrek di Kawasan Banyak Angkrem dan Kedung Kopong, Kecamatan Salaman, Kabupaten Magelang, Jawa Tengah

Banyak Angkrem and Kedung Kopong are medium lowland tropical rain forest areas where many types of orchids can be found. This study aims to determine the types and diversity of orchids found in the area of ​​Banyak Angkrem and Kedung Kopong. Data was collected using the Visual Encounter Survey (VES) method with a radius to the right and to the left 5 m from the observation path with a research area ​​6.24 ha. The results showed that in the two areas found 15 species of orchids consisting of 10 species of epiphytic orchids (Acriopsis lilifolia, Agrostophyllum laxum, Bulbophyllum sp 1, Bulbophyllum sp 2, Dendrobium sagittatum, Dendrobium sp, Oberonia lotsyana, Pholidota sp, Polystaschya concreta, Vanila. planifolia) and 5 species of terrestrial orchids (Geodorum densivelorum, Malaxis ophrydis, Malaxis sp, Nervilia plicata, Perystilus goodyroides). The level of diversity (H ') of orchids in the two regions is classified as moderate with an H' value of 2.22

Habitat Ecology of Epiphytic & Terrestrial Orchids in Langchenphu, Jomotsangkha Wildlife Sanctuary, Bhutan

Orchids are the largest and diverse families of flowering plants. Orchids are found growing mostly in tropical climates affecting various communities socio-economically. Among many protected areas in Bhutan, Jomotshangkha Wildlife Sanctuary (JWS) is the least explored in terms of flora and fauna. The study was carried out to assess diversity, host tree preferences and to determine the growth zone of epiphytic orchids along the altitudinal gradient in Langchenphu gewog in JWS. Transect technique was used in which three trails transect were laid out with 10 sampling plots in each transect. Plots size of 20 m × 20 m along the altitudinal gradients were established maintaining an altitudinal interval of 100 meters between each sample plot. A total of 42 species of epiphytic orchids from 20 genera was recorded of23 host tree species under 15 families were recorded. A significant correlation between diversity of orchid and altitude was observed (r = .927, p < .05). Myrtaceae and Theaceae were the most preferred host tree families (14%) followed by Bignoniaceae (13%) and others respectively. The Study concludes that 57% of total species encountered prefers higher canopy & on a tree with rough bark, Pearson chi-square test (X2(1) = 4.7, p< .05) showed a significant difference between trunk, branches, and canopy. Many epiphytic orchids prefer dense canopy (n = 73, 47%) and living trees (99%). Future studies should be focused more on the flowering season for obtaining better information on orchid diversity in the study area.

An Overview of the Ethnobotanic, Ethnopharmacological and Medicinal Importance of Edible Wild Root Tuber Orchids in Cameroon

Orchids are well known for their beautiful exotic flowers, and food-flavoring products which make them a resource of great economic importance in the global horticultural and food industries. In Cameroon, orchids are particularly important for their tubers which are used for food and traditional medicine. Orchids are therefore of considerable economic importance, traded within and across the regions and out of the country. Few research works have reported for these orchids in Cameroon exploring mostly identification and taxonomy. It is therefore the responsibility and duty of the local population to valorize the tubers for national use based on their awareness of the hidden potentials for food and medicine. Many people will have eaten orchids without realizing, due to the countless products in international trade that contain the seeds of artificially propagated Vanilla orchids. However, this legal trade is only one example of orchids being used as ingredients in food and drink. One example is the trade in chikanda, a cake made from the ground tubers of terrestrial orchids and consumed in several countries in Central and East Africa. Another product made from the ground tubers of terrestrial species is salep, which is used as an ingredient in hot drinks and ice cream and consumed mainly in Turkey and neighboring countries. The high exploitation with no biodiversity and conservation strategic plan by indigenes is a call for concern about the sustainability as future existence of these wild edible orchids is highly threatened; reason why orchids are on the international red list. This paper reviews the ethnobotanical and medicinal uses of orchid roots and tubers with a focus on Cameroon. The review highlights some of the areas that require more focus in terms of research and policy interventions, namely identification of all edible orchids, domestication of edible orchids, and trade controls. Focusing on Cameroon, this paper reviews and examines the edible root and tuber orchids developments based on information from some of the work that has been done in Cameroon, the ethnobotanic, ethnopharmacological and medicinal uses of these orchids.

Asymbiotic seed germination and in vitro seedling development of Orchis militaris, an endangered orchid in Siberia

Background Terrestrial orchids belonging to the Orchis genus are difficult to propagate and are under great pressure in their natural habitats. Studies regarding the influence of photoperiod and temperature regimes on Orchis militaris germination and morphological changes during immature seed development in vitro are scarce. Our aim was to identify photoperiod, temperature, and different nutrient media requirements for optimization of O. militaris seed germination and vigorous seedling production. Results Post-germination morphological changes were recorded with O. militaris seeds collected from 32-day-old fruits, where the percentage of O. militaris seeds without embryo was 38.4%. The highest rate of O. militaris seed germination (82.6%) was obtained on Malmgren modified terrestrial orchid medium (mM), enriched by 5% coconut water, 5% birch sap, and 0.1% AC. Nine percent of seedlings were able to reach the advanced seedling stage (stage 6) after 12 months of maintenance on this medium. In all 3 modified media (Harvais, Knudson С and Malmgren), regeneration was via the production of protocorms and seedlings without callus formation. It was proved that more abundantly vigorous protocorms were formed on the modified Harvais 2 under continuous darkness, while the subculture in Knudson C medium with AC addition could be necessary to stimulate their further development. The regeneration success of the species for in vitro conditions was increased by following its natural seasonal cycle. Conclusion This study demonstrated an efficient micropropagation system for O. militaris using immature seeds and thus widely opened the perspectives for its conservation in nature. The favorable conditions of seed germination periods for in vitro culture, identified as the definite shift of temperatures and photoperiod regimes intrinsic to the species in nature, could improve seedling survival of this medicinally important orchid.