Pelotons visible in the root section of Prasophyllum. [Photo: Emily McQualter]
Prasophyllum aff. validum seedling. [Photo: Emily McQualter]
Prasophyllum aff. validum seedling with leaf. [Photo: Emily McQualter]
Report from an ABRS bursary recipient
Mycorrhizal fungi of Prasophyllum
There are more than 380 orchid taxa in Victoria, at least half of which are threatened. The potential extinction of many of these orchids is largely due to habitat degradation, and destruction caused by agriculture, industrial development and urbanisation. Effective conservation ultimately depends on reintroduction to field sites so as to reinforce depleted populations. For terrestrial orchids, seed germination is the preferred method of propagation as it allows genetic variability to be maintained (Batty et al., 2006).
The genus Prasophyllum currently consists of approximately 80 recognised species in Australia and four in New Zealand (Jones, 1998). Within Australia there are two centres of diversity for the genus: south-western Australia with 25 species (23 endemic) and south-eastern Australia with 50. Within south-eastern Australia approximately 30 species occur in Victoria. Most are threatened and restricted in distribution. Overall, it is one of the least well known of the native orchid genera (Bishop, 1996).
Prasophyllum species are obligate mycotrophic plants that rely on fungi for seed germination. The fungi are also thought to provide nutrients to the adult plants. Current conservation protocols for terrestrial orchids in Australia require propagation with symbiotic mycorrhizal fungi. Unfortunately there is a paucity of knowledge regarding the mycosymbiont of Prasophyllum, hampering conservation and re-introduction efforts. Anecdotal evidence has shown that often the mycorrhizal fungi isolated from adult plants do not germinate seed collected from the same plant. Seed germination trials conducted by the Department of Sustainability and Environment, Victoria, have had no success in germinating Prasophyllum seeds in a range of species. It is possible that the fungi isolated from the species may have been collected at the wrong time of year for protocorms to develop. Therefore, before recovery plans can be implemented for Prasophyllum, basic biological information is required regarding the nature of the mycorrhizal relationship.
This study focuses on two threatened Prasophyllum species: P. aff. validum and P. diversiflorum, both from south-western Victoria. Prasophyllum aff. validum grows in low open grassy heathlands and Prasophyllum diversiflorum (Gorae Leek Orchid) grows along open watercourses and around swamps on heavy black loams.
Underground plant parts were collected for mycorrhizal isolation and Scanning Electron Microscope (SEM) studies. Mycorrhizal fungi were to be isolated from adult plants at four times during 2006: soon after leaves appear following summer, during the period of flower bud growth (winter), while flowering (summer) and as the fruit developed (summer). Due to the current severe drought conditions in Australia, the plants at both populations failed to flower and fruit in 2006. Collections were therefore made at three stages: i) soon after leaves appeared, ii) the period of flower bud growth and iii) dormancy (summer). SEM has been used to determine the location, type and amount of mycorrhizal colonisation.
SEM has demonstrated that the area of fungal colonisation in both species of Prasophyllum during early leaf development occurs in the roots, particularly in the upper root sections. The colonisation occurs primarily in the cortical cells. The fungi enter the orchid through the epidermis and form balls of hyphae known as ‘pelotons’ inside the plant cells. The areas colonised by fungi differ between genera. In Caladenia (Spider Orchid) pelotons are primarily found in the stem-collar region of the plants, in Pterostylis pelotons are found in the underground stem (Ramsay et al., 1986). The morphology of the fungi in both species of Prasophyllum is similar, and the number of cells colonised appears to be unpredictable. According to Warcup (1981), the main fungus associated with Prasophyllum is Ceratobasidium cornigerum, although others occur less commonly.
Fungal isolates from all plants were tested for their ability to germinate seed on oats media with differing sucrose levels. One fungal isolate from P. diversiflorum, isolated at leaf emergence, germinated a low number of P. aff. validum seed. Fungi isolated from plants during flower budding, and those isolated from adult plants of Pterostylis species successfully germinated seed from P. aff. validum. Within two months of the seed germination trials the seedlings developed.
As most mycorrhizal fungi from Australian terrestrial orchids do not sporulate in culture and therefore cannot be identified by normal taxonomic means, DNA from fungal isolates was ITS-sequenced and closest GenBank matches were determined. The effective fungi were morphologically and genetically similar to members of the Rhizoctonia complex and identified as Ceratobasidium. The information gained in this study will provide the basis for further re-introduction and conservation studies.
References
Batty, A.L., Brundrett, M.C., Dixon, K.D. & Sivasithamparam, K. (2006), New methods to improve symbiotic propagation of temperate terrestrial orchid seedlings from axenic culture soil, Australian Journal of Botany 53: 367–374.
Bishop, T. (1996), Field Guide to the Orchids of New South Wales and Victoria, 2nd edn. University of New South Wales Press, Sydney.
Jones, D.L. (1998), Contributions to Tasmanian Orchidaceae— 6: a taxonomic review of Prasophyllum R.Br. in Tasmania, Australian Orchid Research 3: 94–134.
Ramsay, R.R., Dixon, K.W. & Sivasithamparam, K. (1986), Patterns of infection and endophytes associated with Western Australian orchids, Lindleyana 1(3): 203–214.
Warcup, J.H. (1981), The mycorrhizal relationships of Australian orchids, New Phytologist 87: 371–381.