NRCM Silver Jubilee Technical Bulletin (1983-2008) Flammulina velutipes, the culinary medicinal winter mushroom V.P. Sharma Satish Kumar R.P. Tewari National Research Centre for Mushroom (Indian Council of Agricultural Research ) Chambaghat, Solan- 173213 (HP) CONTENTS Page No. 1. Introduction 2. Morphological and molecular characterization 3. History of cultivation 4. World production 5. Nutritional value 6. Medicinal attributes and their properties 7. Physiological requirements 8. Genetics and breeding 9. Preservation of cultures 10. Cultivation in India 11. Cultivation in other countries 12. Effect of environmental factors on the production 13. Post harvest management 14. Insect-pests and diseases 15. Spent mushroom substrate management FOREWORD The land resources in the world for raising food crops are limited and there is no possibility for its further increase. This warrants a wise use of land with due regards to its sustainability for long term productivity. Keeping in view this limitation mushroom cultivation is a wonderful activity which fits very well in the present scenario and also alleviates environmental pollution besides producing a highly valuable protein rich product. India is blessed with varied agro climate, abundance of agricultural waste and manpower making it the most suitable for the cultivation of all types of mushrooms. Our Centre is giving greater emphasis on the diversification in mushroom cultivation. Farmers are being motivated to cultivate these new mushrooms for better returns. Diversification will provide opportunity to the seasonal mushroom growers for round the year cultivation and utilization of different cultivation substrates. I appreciate the efforts and labour put in by the authors in compiling and editing the Bulletin for its use by the mushroom growers and researchers. R.P. Tewari Director National Research Centre for Mushroom, Dated: Chambaghat, Solan- 173 213 (H.P.) INTRODUCTION Varied agroclimatic conditions and availability of agricultural and industrial wastes in India offer great opportunities for cultivating different mushrooms on commercial scale. Introduction of new mushrooms is also important to meet out the increasing public appetite for new and different foods. Total world production of different mushrooms especially Lentinula, Pleurotus, Auricularia and Flammulina has gone up many folds in the last few years. Occasionally the glut of a particular commodity during bumper production period also fetches poor market price to the growers which happened in the early 1990 and made several growers search for alternative crops to supplement their income. Different mushrooms while adding variety, flavour and eye appeal to our food, mushrooms are also considered nutritious because of their proteins, fiber, vitamin and mineral content. A wide variation in the chemical composition of a particular mushroom has been observed by various workers which may be due to the environmental conditions, stage of sampling and method of analysis. Majority of the Indian population being vegetarian, consumption of mushrooms would certainly augument their diet which is deficient in proteins and minerals. Mushroom consumption can thus prove a boon to the growing children as well as breast feeding mothers.Some mushrooms have special significance for medicinal purposes. Specialty mushrooms have been traditionally used in China and Japan for medicinal and tonic purposes since time immemorial.Mushroom consumption has proved beneficial for the patients suffering from hypertension, high sugar and heart problems. Beneficial effects of shiitake consumption are known as enhanced vigour, energy, potency and diminished ageing. Therefore, the cultivation and consumption of mushrooms can contribute towards sound health. Species of Flammulina have been reported to occur naturally on various deciduous tree species namely, poplar (Populus spp), willows (Salix spp), elms (Ulmus spp) plum (Prunus spp), maple (Acer spp) and birch (Betula spp) as a parasite and later as a saprophyte growing on the trunks or stumps of these broad-leaved trees from the end of autumn to early spring. Infection occurs only on wounded or weakened trees. Fruit bodies appear after a few months at temps. between -2 and 14oC and at under low intensity winter light (Poppe, 1974, Zadrazil1999 ).Under in vitro conditions fruit bodies were produced by culturing on potato dextrose agar in darkness at 30o C for 7 days and subsequent transfer to 5oC. (Chandra and Purkayastha,1972). Flammulina velutipes was cultivated as early as 800AD in China and today it is being cultivated all over the world especially in China, Siberia, Asia Minor, Europe, Africa, North America, Australia, Taiwan and Japan. This mushroom is particularly known for its taste and preventive as well as curative properties for liver diseases and gastroenteric ulcers. In addition, winter mushroom has also been reported to contain immunodomodulatory, antitumor and antibiotic substances Common names: Enokitake, Enoki, golden needle mushroom, winter mushrooms, velvet foot, or velvet stem. Taxonomic Position: Kingdom: Fungi;Division: Basidiomycota;Class:Homobasidiomycetes;Order: Agaricales; Family: Tricholomataceae Marasmiaceae;Genus:Flammulina;Species: callistosporioides, elastica, fennae, ferrugineolutea, mediterranea, mexicana, ononidis, populicola, rossica, similes, stratosa, velutipes Morphological and Molecular characterization: Flammulina velutipes is long and thin white mushroom used in the Cuisine of Japan and China. The mushroom naturally grows on the stumps of the Chinese hackberry tree, called enoki in Japanese, but also on some other trees as for example mulberry and persimmon trees. There is a significant difference in appearance between the wild and the cultivated mushrooms. Cultivated mushrooms are not exposed to light resulting in a white color, whereas wild mushrooms usually have a dark brown color. The cultivated mushrooms are also grown to produce long thin stems, whereas wild mushrooms produce a much shorter and thicker stem. Its fruit bodies are small but delicious. The pileus is 2-3 cm in diameter and hemispheric or convex in early stages of its development, gradually opening to a plane as it grows. The surface of the pileus is viscid when moist and is yellowish brown or dark brown, usually with a light brown margin. The flesh is almost white. The gills are white or light-cream colored, and they are adnexed or decurrent to the stipe. The stipe is stiff, 2-9 cm long and 2-8 mm in diameter. The lower part of the stipe is dark brown, while the upper part is light brown and sometimes almost white. The spores are white, with a flat surface, cylinderically oval, and 5-7 X 3-4 µm in size. Neustroeva (1984) studied the morphology of 30 strains of Collybia velutipes collected from 11 tree host species. The strains fell into 2 distinct groups: the 1st group, consisting of isolates collected from linden (Tilia cordata) and elder (Sambucus nigra]) was characterized by more rapid growth. Three strains selected from each group produced fruit bodies within 23-25 days (group 1) and 40-50 days (group 2) after inoculation on a solid substrate. Molecular characterization (Kong et al.,1997), revealed that various isolates of F. velutipes collected worldwide have two differently sized IGR (intergenic region) of 1.61 and 1.58 kb. Based on restriction analysis of the IGR and ITS(internal transcribed spacer) rDNA using 6 different 4-bp-recognizing enzymes, 22 isolates were classified into 4 groups with 92% similarity. In random amplification polymorphic DNA (RAPD) analysis with 20 primers, 68 collected isolates were classified into 7 genotypic classes with 87% similarity. Interestingly, 30 isolates producing white fruit bodies were classified into one group. Nucleotide sequence and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the ribosomal RNA gene (rDNA) regions containing the internal transcribed spacers (ITSs) and the 5.8S rRNA coding sequence was used to differentiate between 7 typical Flammulina strains (MH09201, MH09208, MH09210, MH09234, MH09286, MH09289 and MH09297). These nucleotide sequences revealed the presence of strain-specific deletions, insertions and substitutions (Palapala et al.,2002). RFLP patterns produced using restriction endonucleases DraI, FokI, HaeII, MboII, and NlaIV, enabled identification of specific Flammulina strains. PCR-RFLP analysis of the ITS regions appears to be a useful tool for the identification of Flammulina strains.Digestion of F. velutipes collections with two restriction enzymes, Bgl I and Bst UI, gave variable restriction fragment patterns and revealed biogeographically identifiable patterns. Three haplotypes of F. velutipes were identified with geographical distributions chiefly in Asia, Europe and North America. (Methven et al., 2000). Azuma et al.,1996. A cDNA library was constructed using mRNA from 7-day (7d)-old cultures and also screened for genes that may be involved in fruiting body differentiation. One cDNA clone, FDS (F. velutipes differentiation specific), was isolated by differential screening. History of cultivation: It is reported that (Yang,1986: Wang,1995), F.velutipes first cultivated in China during the 8th century. In 1928, Moriki cultivated it with sawdust and rice bran in Japan (Nakamura, 1981). The mushroom has been cultivated in Japan for over 300 years, initially on wood, and later in the bottles During the 1960s, its cultivation revolutionized in Japan, which became its largest producer in the world and enjoyed this position till the 1980s. Since the early 90's, China has occupied the first place in its production. It was estimated that in the Mainland China its production was about 200,000 tons during 1995 (Meiging, 1997). Production data from different other countries too indicated a faster growth rate in terms of its total production. In the United States, for example, the production of Flammulina increased at an estimated rate of 25% or more per year for the last four years (Royse, 1995). Production of Flammulina is based on synthetic substrate contained in polypropylene bottles or bags. The substrates most utilised are agricultural residues, such as corncobs, cottonseed husk, sugarcane bagasse, etc., besides sawdust (Chang, 1989; Yang, 1986; Fan et al, 1990; Wang, 1995; Royse, 1995). World Production Diversification of the mushroom industry, in terms of number and quantity of species cultivated, has accelerated worldwide during the 1980s and 1990s. Twenty years ago, 70% of the world's mushroom supply was Agaricus bisporus, but by the mid-1990s, this had fallen to only 37%. (Royse, 1997). Total mushroom production worldwide has increased more than 18-fold in the last 32 years, from about 350,000 metric tons in 1965 to about 6,160,800 metric tons in 1997 (Table 1). The bulk of this increase has occurred during the last 15 years. A considerable shift has occurred in the composite of genera that constitute the mushroom supply.During the 1979 production year, the button mushroom, Agaricus bisporus, accounted for over 70 per cent of the world’s supply. By 1997, only 32 per cent of world production was A. bisporus. The People’s Republic of China is the major producer of edible mushrooms, producing about 14 million tons in 2006. Table 1. World production of cultivated edible mushrooms in 1986 and 1997. _________________________________________________________________________ Fresh weight (x 1,000 t) Increase Species 1986 1997 (%) _________________________________________________________________________ Agaricus bisporus 1,227 (56.2%) 1,956 (31.8%) 59.4 Lentinula edodes 314 (14.4%) 1,564 (25.4%) 398.1 Pleurotus spp. 169 (7.7%) 876 (14.2%) 418.3 Auricularia spp. 119 (5.5%) 485 (7.9%) 307.6 Volvariella volvacea 178 (8.2%) 181 (3.0%) 1.7 Flammulina velutipes 100 (4.6%) 285 (4.6%) 130.0 Tremella fuciformis 40 (1.8%) 130 (2.1%) 225.0 Hypsizygus marmoreus — — 74 (1.2%) — Pholiota nameko 25 (1.1%) 56 (0.9%) 124.0 Grifola frondosa — — 33 (0.5%) — Others 10 (0.5%) 518 (8.4%) 5,080.0 Total 2,182 (100.0%) 6,158 (100.0%) 182.2 __________________________________________________________________________ Source: Chang (1999) Table 1. World production of cultivated edible mushrooms in 1978 to 2002. ________________________________________________________________ Year Fresh weight (x 1,000 t) _________________________________________________________________ 1978 10,600 1983 14,510 1986 21,760 1990 37,630 1994 49,093 1997 61,684 2002 122,500 _________________________________________________________________ Source: Chang (2002) Flammulina ranks at fourth place in the category of edible mushrooms for production and consumption. During 1990, its production was estimated to be approximately 143,000 tons, which increased to 230,000 tons in 1994, showing a remarkable jump of 61% (Chang 1996).Worldwide 1986 production of F. velutipes (enokitake) has increased from about 100,000 tonnes in 1986 to about 187,000 t in 1991 (87% increase). Worldwide production of F. velutipes (enokitake has increased from about 143,000 metric tons in 1990 to about 285,000t in 1997 (a 2-fold increase, Chang 1999). Japan is the main producer of winter mushroom (Furukawa 1987). In 1986, Japan produced 74,387tonnes; by 1991, production had risen to 95,123 tones and, by 1997, Japan produced 174,100 tones -an increase of about 45% in six years. From these data, it is evident that other countries are enjoying a faster growth rate, in terms of total production. In the United States, for example, winter mushroom production has increased at an estimated rate of 25% or more per year for the last four years. Lee and Park (1994) investigated the profitability of bottle mushroom cultivation in the Korea Republic and suggested that for a production level of 2000 bottles per day, it requires a total expenditure of worth 5 million and obtains an annual net income of worth 110 million. For a production level of 1000 bottles per day, Pleurotus ostreatus requires a total expenditure of worth 300 million and provides an annual net income of worth 30 million. Nutritional value: Flammulina velutipes is a popular edible mushroom in Japan where a tender cultivated form of the wild version is called "enokitake." It is commonly used in Japanese cooking and increasingly can be found in salads in gourmet restaurants. F. velutipes contains 31.2 per cent protein, 5.8 per cent fat, 3.3 per cent fiber and 7.6 per cent ash on the basis dry material. Medicinal attributes and their properties Flammulina velutipes, a delicious mushroom is rich in peroxidase, superoxide dismutase, and others and can prevent some severe diseases like cancer and coronary heart disease. It is beneficial for physical and intelligence of children. Winter mushroom contains compounds that prevent as well as cure liver disease and gastroenteric ulcers provided it is taken on a regular basis (Ying et al., 1987; Yoshioka et al., 1973). In addition, like many other specialty mushrooms, winter mushroom contains immunodomodulatory, antitumor, tumor inhibiting, and anti-biotic substances. Both mycelium and fruitbody of F. velutipes could be recommended for formulating antioxidative dietary supplements. Winter mushroom contains several types of amino acids including valine, which inhibits the growth of Ehrlich ascities tumour and sarcoma 180 in mice; and lysine. It also reported to increase body height and weight (Ying et al., 1987). Winter mushroom contains a cardiotoxic protein (flammutoxin) that is rendered harmless when subjected to heat (100 degrees C) for 20 minutes. The dose is 8-9 g/day. Purification of lysine and flammutoxin and revealed that it is a single polypeptide chain of Mr 32 000 and pK about 5.4 (Bernheimer and Oppenheim,1987). It contains large amounts of tryptophan, serine and glycine, and few or none of the sulphur- containing amino acids. The toxin appears not to be a phospholipase and it was not inhibitable by any of a variety of lipids. Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. Flammutoxin did not lyse human erythrocytes in the presence of non-electrolytes with hydrodynamic diameters of >5.0 nm, although it caused leakage of potassium ions and swelling of the cells under the same conditions(Tomita et al., 1998). Flammutoxin, assemble into a pore- forming annular oligomer with outer and inner diameters of 10 and 5 nm on the target cells. The electrophysiological properties of flammutoxin channels using planar lipid bilayer technique were studied, and found that the larger-conductance channel of flammutoxin corresponds to the haemolytic pore complex, while the smaller-conductance channel may reflect the intermediate state(s) of the assembling toxin(Gulnora et al., 2000) . Cao et al. (1989) reported that PA3DE shown to have a MW of 5.4 x 106 which contained D-glucose, D-mannose and L-fucose in the molar ratio 22.31:1.46:1.00, and had β -glycosidic linkage. It showed inhibitory activity against the Sarcoma 180 tumor in mice. A new fungal immunomodulatory protein (FIP-fve), having molecular mass of FIP-fve determined by SDS-PAGE agreed well with the value of 12 704 Da calculated