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Specialty Gourmet and Medicinal Fungi Research in Tasmania


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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002

UAEM. ISBN 968-878-105-3


Specialty Gourmet and Medicinal Fungi Research in Tasmania
K.G. Stott1, W. Gill1, 2, C. Mohammed1a and M. Brown2

1Specialty Gourmet and Medicinal Fungi Research, Tasmanian Institute of Agricultural Research, 13 St. Johns Avenue, New Town, Tas, 7008, Australia. <Karen.stott@dpiwe.tas.gov.au>

2 Huon Valley Mushrooms, Main Road, Glen Huon, Tasmania, 7109, Australia

Abstract
In 1999, to meet the needs of industry and research in Australia, an important research facility for specialty gourmet and medicinal fungi was established in Tasmania. This premier research facility also provides education in mycology and specialty mushrooms, via on-line courses through the University of Tasmania. Current research projects investigate the edible fungi: Grifola frondosa (Maitake), Morchella species (Morel) and Tricholoma matsutake (Matsutake).

The Japanese bag method was used to assess the response of maitake to eucalyptus substrate ameliorated with different percentages of rice bran, maize meal and wheat bran (10%, 20%, 30%). The response of maitake to these substrates indicates that only relatively small quantities of additive will be required for successful cultivation.

The edible morel fungus was collected in Tasmania, in forests, in garden soil, in bark chip mulch, and in other locations. On the basis of their morphological characters, fruit bodies were placed into one of the three following species: M. elata, M. deliciosa, M. esculenta var. crassipes/angusticeps. The sclerotial production of Tasmanian and overseas isolates was compared.


Studies initiated in the year 2000 to elucidate the physiology and morphology of T. matsutake mycorrhizas with the view to cultivating the fungus saprophytically, are briefly described.

Introduction
In Australia, the mushroom industry is worth US$ 100 million. In Tasmania, it is valued at US$ 2.5 million and expected to double within two years. This is largely due to increasing interest in specialty mushrooms, of which Australia imports 50-100t annually. Research in Tasmania seeks to optimise existing cultural techniques for specialty mushrooms such as maitake Grifola frondosa using cheap, locally sourced substrate materials. More fundamental research is focusing on specialty mushrooms such as morel Morchella spp and matsutake Tricholoma matsutake that are either very difficult to cultivate or have never been artificially cultured.
Maitake is well known as an Asian edible fungus which has both nutritional and medicinal value and can fetch US$ 20 per kilogram on the fresh market. It is sold in various forms: fresh, dried, capsules and tea (Stamets 1993, Royse 1997). Internationally the cultivation of maitake is based on hardwood sawdusts such as oak, poplar, cottonwood, elm, willow and alder (Stamets 1993). In Australia these substrates are unavailable, but Eucalyptus sawdust provides a readily available, low cost substrate for cultivation. Suggested supplements to sawdusts are wheat bran, rice bran, oat bran and maize meal (Stamets 1993, Kirchhoff 1996, Mayuzumi and Mizuno 1997). In Tasmania, amended Eucalyptus sawdust is commonly used for the cultivation of shiitake Lentinula edodes. We have therefore been determining optimal additive levels and substrates to supplement Eucalyptus sawdust for the production of maitake.
Morels, harvested from nature, are currently sold as gourmet mushrooms in major Australian wholesale markets for up to US $30 per kilogram (Smedley, P. 2001, pers. comm.). In 1988, a US patent was granted for the cultivation of the white morel (M. esculenta) under controlled conditions (Ower et al. 1988). A company holding the patent is currently commercially cultivating white morels. Unfortunately, others have achieved very limited success in obtaining fruiting of morels under controlled conditions.
A stage of the morel life cycle not present in other cultivated mushrooms is sclerotium formation. The sclerotium of the morel is a relatively large structure (1-50 mm diameter) composed of aggregated mycelium - large cells with very thick walls that allow the fungus to survive adverse natural conditions, such as winter. During spring, in nature, the sclerotium has two options for germination: to form new mycelium or to produce a fruiting body. Very specific conditions of nutrition, humidity, carbon dioxide levels and temperature must be met for fruiting initials or primordia to form from sclerotia and for these to develop into fully mature fruit bodies. Understanding sclerotial formation and development is crucial in any attempt to fruit morel isolates.
Sclerotia are known to be stimulated when mycelium grows from a nutrient-poor substrate to a nutrient- rich substrate (Ower et al. 1986, 1988, Volk and Leonard 1989). Since prolific large sclerotial formation must be directly related to fruit body production several researchers have investigated sclerotial production under the effect of successive growth on nutrient-poor and nutrient-rich media in split plates (Amir et al. 1992, 1993, Philippoussis and Balis 1995, Faris et al. 1996). Results are sometimes contradictory; sclerotial formation has been reported on both nutrient-rich and nutrient-poor sides or only on the inoculum side, whatever the medium inoculated.
Buscot (1993) observed that two types of M. esculenta sclerotia formed on two different solid media, Buscot A and Buscot B: "early encrusting sclerotia" ("EES") and "late isolated sclerotia" ("LIS"). The production of "EES" was described as occurring when mycelial growth was interrupted physically by the edge of the petri plate, as was shown by using different sized petri plates. Whilst the production of "EES" could occur in response to physical interruption, it could also be a response to nutrient supply or change in turgor pressure of media at the interface between media and petri plate. The initiation of "LIS" appears to be associated with the age of the mycelial culture as a few isolated sclerotia form, after the "EES" stage, anywhere on the mycelium, irrespective of the size of the petri plate. It has been suggested that "LIS" are similar to mycelial structures found in connection with ascocarps in nature, due to their similar temperature requirements, cold resistance and biochemical properties (Buscot 1993) but this has not been proven.
Tricholoma matsutake (Ito et Imai) Sing. (Matsutake) is the premier fungal delicacy of Japan, fetching in excess of US $1000 per kilogram fresh weight on the Japanese market. However, in the past few decades, there has been a dramatic decrease in the availability of the fungus.
Despite nearly a century of research, with the exception of sporadic unsubstantiated reports of single fruit body formation (Hall and Wang 1998), attempts to establish a truly artificial matsutake cultivation system have proven unsuccessful. In contrast, Joseph Talon first achieved the artificial cultivation of the ectomycorrhizal Périgord black truffle in the early 1800s. By transferring oak seedlings growing near truffle-bearing trees and planting them out, he found that the new seedlings would also produce truffles. Although modern methods have improved on this technique, the basic principles of infecting and outplanting seedlings are the same. Attempts at cultivating Matsutake by this method have failed. Following initial infection, T. matsutake became pathogenic and the host seedlings died.
One of the prerequisites to progressing the development of any successful cultivation technique must therefore be an understanding of the interaction between T. matsutake and host tree roots. While originally considered an ectomycorrhizal association, the interaction has since been variously described as pathogenic and symbiotic. It was not until recently, however, that the T. matsutake-host interaction was confirmed as ectomycorrhizal (Gill et al. 1999, Yamada et al. 1999). Subsequent work has indicated that morphologically (Gill et al. 2000) and in culture (Guerin-Laguette et al. 2000), T. matsutake possesses characteristics atypical of ectomycorrhizal fungi. It is the atypical fungus-host interaction and the cultural characteristics of the fungus that are being investigated in order to develop an artificial cultivation system for this highly prized edible Japanese mushroom.

Materials and Methods
The Japanese bag method (Lee 1996, Mayuzumi and Mizuno 1997, Royse and Guardino 1997) was used to assess the response of maitake to Eucalyptus sawdust which is of a fine texture and economical to the grower. Substrate was ameliorated with different percentages (10%, 20%, 30%) of rice bran, maize meal and wheat bran by dry weight. Bags containing one kilogram of substrate were autoclaved for two hours at 120oC and allowed to cool before inoculating with strains C200 (Fungi Perfecti, USA), M74 (Chinese University of Hong Kong) and WC808 (Pennsylvania State University, USA). Eight replicates of each strain for each additive treatment plus a control with no additive were prepared. Colonisation occurred in the dark at a temperature of 25oC during spawn run. The rate and area of colonisation was determined by measuring, on a weekly basis, a defined area of each 1 kg log. Data was analysed using Generalised Linear Mixed Model Analysis (Genstat 5, Release 4.1, Lawes Agricultural Trust).
Morel fruit bodies were collected in Tasmania in spring 2000. Accession details of collections were recorded with specimens photographed and dried for future study.
Vegetative cultures of seven Tasmanian morel fruit bodies (representative of the three different species found in Tasmania) and imported isolates of M. angusticeps (Fungi Perfecti, USA), M. esculenta (INRA, France) and M. hortensis (INRA, France) were selected for the trial to investigate sclerotial formation under the effect of successive growth on nutrient-poor (distilled water agar; DWA) and nutrient-rich (malt extract agar; MEA) media in split plates. Only the Tasmanian isolates were grown on Buscot’s A and B media (Buscot 1993) to determine "EES" and "LIS" sclerotial formation.

Standard complex mycorrhizal culture media were examined for their ability to support and promote T. matsutake growth. Pachlewski’s (Pachlewski and Pachlewski 1974), Ohta’s matsutake and Ohta’s ectomycorrhiza (OhtaM and OhtaE respectively, Ohta 1977) and modified Melin Norkrans (MMN, Marx 1968) agar in 90 mm Petri dishes were inoculated in the centre with 5mm plugs of eight strains of T. matsutake from the growing margin of a colony growing on MMN. Each strain was inoculated onto three plates of each medium as replicates. Plates were sealed and incubated at 25ºC in darkness. The bottom of each plate was marked with two perpendicular centre lines and each developing colony was measured at three-day intervals along the four radii.


In a preliminary investigation, Tasmanian coniferous tree species, Pseudotsuga menziesii, Pinus pinaster and Pinus radiata, were tested as possible hosts for mycorrhization by matsutake. Initially, seeds of each species were washed and sterilised following a standard procedure (Guerin-Laguette et al. 2000). The seeds were then transferred to potato dextrose agar (PDA) in petri dishes and

germinated at 25ºC in either 24 hour darkness or 24 hour light. Germination rate and seedling elongation rate were assessed to select the best tree species for mycorrhization.


Seeds of the selected tree species were then subjected to different washing, sterilisation and incubation treatments to optimise the germination and elongation rates and the rate of production of short lateral roots, a requirement for mycorrhization.

Results
Initial data indicated that maitake isolates rapidly colonised Eucalyptus sawdust with additives of 20% wheat or rice bran and 30% maize meal. Analysis of data at the end of the trial found that 10% rice bran, 20% wheat bran or 20% maize meal encouraged the greatest area of hyphal colonisation for two of the three isolates tested, WC808 and M74 respectively, which took between 33-79 days to fully colonise substrate.
Thirty one morel collections were made in a range of Tasmanian habitats, from forest to garden soil and bark mulch. Twenty six vegetative cultures were successfully isolated and stored at the Tasmanian Institute of Agricultural Research (TIAR). On the basis of morphological characters fruit bodies were placed into one of the three following species: M. elata, M. deliciosa, M. esculenta var. crassipes/angusticeps.
Sclerotia deemed similar to Buscot's "EES" (ie. at the edge of the plate or around the inoculum) were, in Tasmanian morel isolates, formed at the same time as sclerotia which were visually comparable to Buscot's "LIS" (ie. all over the plate), whatever the type of Buscot medium (A or B).
In split plates Tasmanian isolates produced sclerotia on DWA, irrespective of whether MEA or DWA was inoculated. By contrast, imported isolates of M. esculenta and M. hortensis only formed sclerotia on MEA irrespective of whether MEA or DWA was inoculated. M. angusticeps did not form sclerotia when inoculated on DWA but sclerotia formed on both DWA and MEA when MEA was inoculated.
There were no marked differences between the size and quantity of sclerotia formed by the different Tasmanian isolates on either Buscot media or DWA.
Of the eight matsutake strains tested, five preferred OhtaE and one strain favoured OhtaM medium. The remaining two strains grew aberrantly and the results were inconclusive. OhtaE was therefore selected as the medium of choice for laboratory maintenance of T. matsutake. From this test, three strains identified as the most vigorous were selected as test organisms for subsequent culture studies.
From the preliminary trial, P. radiata showed the greatest germination and elongation rates in darkness and was selected as the host species. Subsequent treatment trials demonstrated that a wash in cold running water followed by sterilisation in 30% hydrogen peroxide and incubation on PDA in darkness at 25ºC induced the greatest rate of germination and seedling elongation. However, P. radiata did not readily produce lateral roots. Attempts at root initiation indicated that P. radiata seedlings required undercutting and hormone treatment to induce formation of short laterals.

Discussion
Sawdust plus 20% wheat bran or rice bran has been recommended as suitable substrate for the cultivation of maitake (Takama et al. 1981, Lee 1996, Royse 1996). This study found the addition of 10% rice bran, 20% wheat bran or 20% maize meal to Eucalyptus sawdust encouraged hyphal colonisation. This agrees with Kirchoff (1996) who found the addition of 20% corn meal or 10% wheat bran to beechwood sawdust encouraged primordia. In Tasmania, wheat bran would be favoured as an additive because of its availability and cost. Further work with the Eucalyptus media plus wheat bran is necessary to determine optimal environmental conditions for subsequent stages of the maitake life cycle involved in the cultivation of this edible fungus in Tasmania. Our research indicates that Eucalyptus sawdust may be adapted to grow a wide range of saprophytic specialty mushrooms. The use of this economical and readily available material will allow expansion and diversification of the specialty mushroom industry in Tasmania.

The characteristic production of "EES" and "LIS", as per Buscot (1993), was not observed with any of the seven Tasmanian morel isolates belonging to three species. However it should be noted that in his studies of "EES" and "LIS" formation, Buscot only used isolates from a very narrow genetic source (mono or polyspore isolates from a single fruitbody). Our work indicates the importance of screening isolates from a large pool of genetic material in order to detect systematically different behaviour in sclerotial formation.


Tasmanian morel isolates only produced sclerotia on DWA irrespective of whether an isolate was inoculated on the MEA or DWA of the split-plates. This agrees with Faris et al. (1996) who found Australian morel isolates (species not mentioned) on split-plates with nutrient-poor carnation leaf agar (CLA) and MEA, produced sclerotia on CLA irrespective of whether CLA or MEA had been inoculated. Our result is also partially supported by the use of split-plates containing Noble agar (nutrient-poor) and PDA (nutrient-rich) to study sclerotial production (Amir et al. 1992, 1993). Noble agar was inoculated with M. esculenta and sclerotia initially formed on Noble agar but 12 h later also formed on PDA (Amir et al. 1992).
We consider that until Buscot's "EES" or "LIS" observations of sclerotial formation are further investigated with a wider range of morel isolates that the split plate technique for screening an isolate's ability to produce sclerotia is more reliable for the comparative study of sclerotial formation in morels.
Our preliminary studies showed that the sclerotial response of Tasmanian isolates studied (M. elata, M. deliciosa, M. esculenta var. crassipes/angusticeps) appears different to that of imported M. angusticeps, M. esculenta and M. hortensis. Our results reflect the taxonomic confusion and variability within this genus and highlight the difficulties experienced when trying to develop an understanding of the nutritional and physiological requirements necessary to encourage sclerotial production. Current work comparing a larger number of Australian morel isolates with a wider range of international isolates is in progress.
As do other ectomycorrhizal fungi in pure culture, matsutake grows very slowly. Producing sufficient inoculum to initiate mycorrhization processes is a very time-consuming task. The need to identify the medium which promotes the most vigorous growth is paramount, particularly when considering future commercialisation strategies. The identification here of OhtaE as the medium of preference is unusual in that Ohta (1990) created a defined medium specifically for matsutake (OhtaM) which, according to Ohta, outperformed OhtaE as a laboratory medium. While matsutake growth on OhtaE in pure culture is greater than other standard complex mycorrhizal media, there is a requirement for further refinement of a medium to optimise the growth rate and hyphal vigour.

While the natural primary host species of matsutake is Pinus densiflora (Japanese red pine, akamatsu), matsutake has been reported in association with a number of tree species (Masui 1927, Trappe 1962, Tominaga 1971, Ogawa 1976). As P. densiflora seed is not readily available in Tasmania, other coniferous species were tested as potential matsutake host plants, given the apparent non-host specificity of Matsutake. Pinus radiata seed produces a vigorous, rapidly growing seedling which can be stimulated artificially to produce necessary short lateral roots. This result concurs with Hall and Wang (1998) who recommend P. radiata as the host species for artificial mycorrhization with Matsutake. However, there is little information available regarding lateral root formation. This work identified the parameters required to produce healthy seedlings with abundant lateral roots in as little as three weeks from seed. This is important not only for establishing mycorrhizas for further physiological and morphological investigation, but is also advantageous commercially. Considering the atypical behaviour of matsutake, tree species outside the Pinaceae will be screened for their potential as host plants.


Despite the huge commercial potential of matsutake and nearly 100 years of research, little is known about the physiology and morphology of T. matsutake mycorrhizas. Basic cultural and interaction studies are still required in order to understand the atypical behaviour of T. matsutake and to develop an artificial cultivation system based on that understanding. By establishing mycorrhizal symbioses on appropriate host tree roots, the interaction can be investigated, characterised, and ideally, controlled.
In Australia, as in other countries worldwide, there has been a steady decline in the teaching of mycology at academic institutes. Mycological institutes are diminished in terms of scope, personnel and available financial resources. All this has been occurring at a time when the volume and monetary value of the specialty mushroom industry is steadily expanding (Chang 1996, Royse 1997). As market demand and production increases, the need for trained staff by both the mushroom industry and those research organisations that support industry, will grow. The research group at the Tasmanian Institute of Agricultural Research will not only act as the main research provider for the specialty mushroom industry in Australia but also, in collaboration with the University of Tasmania, provide an on-line residential course on specialty gourmet and medicinal fungi tailored to the needs of Australians but also relevant to participants from other countries.

Acknowledgements
This work has been made possible by the financial support of the Rural Industries Research and Development Corporation, the Department of Industry Science and Resources and Huon Valley Mushrooms. The commitment of the Tasmanian Institute of Agricultural Research and the University of Tasmania to the establishment of the Specialty Gourmet and Medicinal Fungi Research Group is gratefully acknowledged.

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