Mushrooms Diversity of Amrite Community Forest, Kapilwastu District, Western Nepal‖ Has Been Carried out Under My Supervision

MUSHROOMS DIVERSITY OF AMRITE COMMUNITY

FOREST, KAPILWASTU DISTRICT, WESTERN NEPAL

A DISSERTATION SUBMITTED FOR THE PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE MASTER DEGREE IN BOTANY

By Bhojraj Pokhrel Symbol No. : 13034 (2067/68) T.U. Regd. No. : 5-2-50-1257-2002

Central Department of Botany Tribhuvan University Kirtipur, Nepal April, 2017

RECOMENDATION

This is to certify that Mr. Bhojraj Pokhrel has carried out the dissertation work submitted for the partial fulfillment of M.Sc. degree in Botany, entitled ―Mushrooms Diversity of Amrite Community Forest, Kapilwastu District, Western Nepal‖ has been carried out under my supervision. To the best of my knowledge, this entire work is based on the results of his work and results have not been used or submitted in any other academic degrees. I, therefore, recommended this dissertation to be accepted for partial fulfillment of Masters Degree in Botany from Tribhuvan University.

______Dr. Chandra Prasad Pokhrel (Supervisor) Central Department of Botany Tribhuwan University Kirtipur, Nepal

Date: 29/12/2073

LETTER OF APPORVAL

The dissertation paper entitled “Mushroom Diversity of Amrite Community Forest, Kapilwastu District, Western Nepal’’ submitted at the central department of botany, Tribhuvan University by Mr. Bhojraj Pokhrel has been accepted for a partial fulfillment of Master's of Science in Botany (Plant Pathology).

EVALUATION COMMITTEE

...... Dr. Chandra Prasad Pokhrel Prof. Dr. Mohan Siwakoti (Supervisor) (Head of Department) Central Department of Botany Central Department of Botany Tribhuvan University, Kathmandu, Nepal Tribhuvan University, Kathmandu, Nepal

...... Dr. Jay Kant Raut Dr. Hari Prasad Aryal (External Examiner) (Internal Examiner) Senior Scientific Officer Central Department of Botany Nepal Academy of Science and Tribhuvan University, Kathmandu, Nepal Technology (NAST), Khumaltar, Lalitpur

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ACKNOWLEDGEMENT

I would like to express my sincere gratitude to my respected supervisor Dr. Chandra Pokhrel who guided me giving suggestion and encouraged for my research work. His fruitful discussion always kept on sharpening my ideas while working under him.

It‟s my pleasure to express my sincere gratitude to Dr. Ganesh Neupane and Dr. Sanjay Kumar Jha, Central Department of Botany, Tribhuvan University for their Conscience effort, guidance, valuable suggestions, and cooperation in completion of my dissertation.

My special and warmful thanks go to Dr. Hari Prasad Aryal, Central Department of Botany, Pathology uint, Tribhuvan University for his consistent guidance, suggestions and helpful support for the identification of unidentified mushrooms.

I would also like to express my heartfelt thanks to the Head Prof. Dr. Mohan Siwakoti of Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, for providing the essential materials for my work.

I am thankful to Water Engineering and Training centre Pvt. Ltd. for providing laboratory facilities to accomplished nutrient analysis of mushrooms for my dissertation work.

I would like to thank my friends Ghanshyam Chaudhary, Surya BC and all other teaching and non-teaching staffs of Central Department of Botany, Kirtipur who supported in my work.

I would like to express my thanks to Mr. Dhruv Prasad for his unremitting contribution in collection of mushroom, providing GPS data, computer typing and setting and providing valuable suggestion to accomplish this dissertation work

Last but not the least, I am very much grateful to all my family members whose regular encouragement and inspirations helped me a lot in the completion of this work.

- Bhojraj Pokhrel

LIST OF ABBREVIATIONS

% - Percent

µ - micron oc - Degree Celsius

Alt. - Altitude

AOAC - Association of Official Analytical Chemists

APHA -American Public Health Association

CDB, TU - Central Department of Botany, Tribhuvan University

CN - Collection number

Fig. - Figure

Kg - kilogram mg - milligram mL - milliliter

Spp. - species

SPSS - Statistical Package for Social Science

ABSTRACT

The goal of study was to document the constituents of mushroom and chemical analysis of two edible species of mushroom one wild edible and other cultivated edible, that are found in Amrite Community Forest of Banganga Municipality and its vicinity, Kapilwastu district, Western Nepal.

The present study was conducted during the year 2016 from June to September in the Amrite Community Forest of Banganga Municipality-17. The different species of mushrooms were collected. During the collection altogether 38 wild mushroom specimens were collected. Among them 34 species were identified up to generic level belonging to 16 families. Coprinaceae was the largest famiy and Coprinus species were the dominant species in the study site. The study area is Shorea robusta dominant forest. On the basis of habitat, 24 species were found growing on the soil, 7 on the wood, 4 on the animal dung, 2 on tree trunk and 1 on rock.

Ethomycological study was done with the local people. According to them the mushrooms which are colorful and have pungent and bad smell are poisonous. The local people (Tharus) go for the collection of mushroom like Scleroderma species and Termitomyces species. Some local people used the mushroom for the medicinal purposes. The mushrooms like Auricularia auricular, Scleroderma spp. and Pycnoporus cinnabarinus were used as medicinal purposes. Only few local people have knowledge of medicinal value of mushroom.

Nutrient analysis of cultivated edible and wild edible species revealed that Fe and Ca were found higher in wild edible species (Scleroderma cepa) while Mn, Mg, Na, K, Phosphate and protein content were found comparatively higher in cultivated edible species (Pleurotus ostreatus).

Table of Contents

Contents Page

Recommendation i i

Letter of Approval i i i

Acknowledgement iv

List of Abbreviations v

Abstract vi

1 Introduction 1-10 1.1 Background 1 1.1.1 Concept of mushroom and toadstool 2 1.1.2 Essential parts of mushroom 3 1.1.3 Habitat and Ecology 5 1.1.4 Mushroom Poisoning 5 1.1.5 Nutritional and Medicinal value 6 1.1.6 Ethnomycological aspect 9 1.2 Objectives, Justification and Limitations 10 1.2.1 Objectives 10 1.2.2 Justification 10 1.2.3 Limitations 10 2 Literature Review 11-15 3 Materials and Method 15-20 3.1 Study Area 15 3.1.1 Physiography 15 3.1.2 Climate 17 3.2 Materials and equipments 18 3.3 Chemicals 18 3.4 Preservation of mushrooms 19

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3.4.1 Dry preservation 19 3.4.2 Liquid preservation 19 3.5 Microscopic study 19 3.6 Spore print 19 3.7 Identification of mushrooms 19 3.8 Nutrient analysis 20 4 Result 36-49 4.1 Mushoom Composition 36 4.2 Detailed studies on some collected species 40 4.3 Nutrient analysis 46 4.4 Indigenous knowledge and therapeutic use 49 5 Discussion 50-52 6 Conclusion and Recommendation 53 6.1 Conclusion 53 6.2 Recommendation 53 References 54-64 Annexes 65-67 Photoplates 68-70

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List of Tables Table 1: List of mushrooms collected from the Amrite community forest, 35 Banganga-17, Kapilwastu. Table 2: Nutrient Analysis of wild edible (Scleroderma sepa) and 46 cultivated edible(Pleurotus osteratus) through Atomic Absorption Spectrophotometer and UV visible Spectrophotometer for phosphate analysis.

List of Figures

Figure 1: Fruiting body of mushroom (Source: https: // www. google. com. np/ ) Figure 2: Map of Kapilwastu district showing Banganga municipality 16 Figure 3: Map of Amrite community forest, Banganga-17, Kapilwastu 16 Figure 4: Ombrothermic representation of climate in the study area 17 (Kapilwastu) from 2013-2016. (Source: Metrological department, Bhairahawa) Figure 5: Number of species with respect to families. 38 Figure 6: Distribution of mushroom on the basis of their habitat. 39 Figure 7: Graph showing nutritional variation in wild and cultivated 47 edible species. Figure 8: Pie chart showing protein concentration (%) in wild edible and 47 cultivated edible species.

CHAPTER ONE INTRODUCTION

1.1 Background Mushroom are seasonal fungi which occupy diverse niche in nature in forest ecosystem. They predominantly occur during the rainy season and also during spring when the snow melts. Mushrooms are 'fruiting body' of fungal mycelium. They are macromycetes forming macroscopic fruiting bodies such as agarics, boletes, jelly fungi, coral fungi, stinkhorns, bracket fungi, puffballs and bird‟s nest fungi. They are fleshy, subfleshy, or sometimes leathery and woody and bear their fertile surface either on lamellae or lining the tubes, opening out by means of pores. There are numerous fungi that produce fleshy fruiting bodies commonly known as mushrooms belonging to group Ascomycetes and Basidiomycetes. Mushrooms can be classified as three categories by their tropic pattern; saprophytes, parasites and mycorrhizae. The most commonly grown mushrooms are saprophytes, decomposers in an ecosystem growing on organic matters like wood, leaves and straw in nature. Macro fungi are heterotrophic saprotrophs and utilize lingo cellulosic wastes. The organic materials from which macro fungi derive their nutrition are referred to as substrates. They are natural scavenger which convert the dead organic matter into protein, minerals and alkoloids. Nepal possesses diverse phytogeographical zones related to altitude and other factors. Thus the vegetation varies greatly from east to west and from north to south. These varied elements have enriched Nepal with economically important mycoflora (Adhikari, 1988a). Macro fungi may be edible, inedible, medicinal and poisonous. Many kinds of macro fungi are not edible, but also possess tonic and medicinal properties (Chang & Miles, 2004).There are some mushrooms which are domesticated and grown for commercial purpose but many edible mushrooms are still wild in the forest. They are not only a good source of nutrients and medicine but also function in nutrient recycling and act as a niche for several animal resources.

Ethnomycological studies show predictable differences between populations primarily due to cultured traditions. Mushrooms are much more used by some tribes than other, even within mushroom eating tribes, personal differences exist. Some of the differences are linked to religious belief, others to the surrounding dominant

vegetation type or to the combination of several factors. Cultivation of macromycetes in negligible for the moment and the sporophores are simply picked in nature. The trible people live in consonance with nature are heavily dependent on forest resources for their livelihood. Though, they are settled in forests, the basic instinct of hunting in the wild for their food by various means by hunting, collecting, domesticating and purchasing in the nearby villages. With the present soaring cost of meat and the tribal people are hunting mushrooms, usually in close proximity to their inhabited areas in forests as an alternative source of protein.

To date about 1271 species of macrofungi belonging to 357 genera and 108 families of mushrooms are recorded from Nepal (Adhikari, 2014). Out of these, 228 species have food value (Christensen et al. 2008), while 89 species are medicinal and 100 species poisonous (Adhikari, 2014; Aryal and Budathoki, 2014).

Wild edible mushrooms (WEMs) are important contributions to rural and tribal livelihood (Christensen et al., 2008). For many years various macrofungal species have been used worldwide in preparing dishes with high protein and mineral content. Despite this, WEMs are seldom included in valuation of tropical forests, which has traditionally been mostly based on a financial appraisal of timber stock. WEMs are an important and major component, performing roles both in subsistence use and economic generation for forest fringe communities. These have been overlooked partly due to the lack of knowledge about the resource quantities available and suitable inventory or survey methods to initially assess resource status(Baker, 2000) as the production of WEMs may vary from year to year (Cai et al., 2011).

1.1.1 Concept of mushroom and toadstool The general belief that one or a few species of mushroom are safe to eat and had lead to the concept of mushroom and toadstool to denote edible and poisonous fungi respectively.

The term mushrooms and toadstools are generally applied on those species which are fleshy gill bearing agarics species that are either edible or poisonous. Mushrooms and toadstools both resembles with each other very closely so there is no exact rules and tests which have been found to distinguish or differentiate morphologically (Snell & Dick, 1971).

Miller (1984) defined the mushroom as the term applied to both the poisonous and edible species of agarics as gilled mushroom. Conversely, Purkayastha and Chandra (1985) pointed out that agaricus or other group of fleshy species of fungi are recognized as 'mushroom' which may be edible, inedible, poisonous or non- poisonous.

The word 'toadstool' is derived from the German word 'toad stuhl' which means death- stool causing sickness and even death. The poisonous members however known as toadstool.

Kibby (1979) defined toadstool as inedible or poisonous groups. The toadstool are also gill bearing agarics group but mostly inedible and poisonous. The 'Dictionary of Botany' defined toadstool as synonymous with the mushroom in both the narrow and broad sense, among them very few are edible but is more often used for inedible species. Pacioni (1985) made a clear distiction between mushroom and toadstool considering only the edible species and inedible species respectively.

1.1.2 Essential parts of mushroom

A mushroom develops from a nodule, or pinhead, less than two millimeters in diameter, called a primordium, which is typically found on or near the surface of the substrate. It is formed within the mycelium, the mass of threadlike hyphae that make up the fungus. The primordium enlarges into a roundish structure of interwoven hyphae roughly resembling an egg, called a "button". The button has a cottony roll of mycelium, the universal veil, that surrounds the developing fruit body. As the egg expands, the universal veil ruptures and may remain as a cup, or volva, at the base of the stalk, or as warts or volval patches on the cap. Many mushrooms lack a universal veil, therefore they do not have either a volva or volval patches. Often, a second layer of tissue, the partial veil, covers the bladelike gills that bear spores. As the cap expands, the veil breaks, and remnants of the partial veil may remain as a ring, or annulus, around the middle of the stalk or as fragments hanging from the margin of the cap. The ring may be skirt-like as in some species of Amanita, collar-like as in many species of Lepiota, or merely the faint remnants of a cortina (a partial veil composed of filaments resembling a spiderweb), which is typical of the

genus Cortinarius. Mushrooms lacking partial veils do not form an annulus (Stuntz, 1972) .

The stalk (also called the stipe, or stem) may be central and support the cap in the middle, or it may be off-center and/or lateral, as in species of Pleurotus and Panus. In other mushrooms, a stalk may be absent, as in the polypores that form shelf-like brackets. Puffballslack a stalk, but may have a supporting base. Other mushrooms, such as truffles, jellies, earthstars, and bird's nests, usually do not have stalks, and a specialized mycological vocabulary exists to describe their parts.

The way the gills attach to the top of the stalk is an important feature of mushroom morphology. Mushrooms in the genera Agaricus,Amanita, Lepiota and Pluteus, among others, have free gills that do not extend to the top of the stalk. Others have decurrent gills that extend down the stalk, as in the genera Omphalotus and Pleurotus. There are a great number of variations between the extremes of free and decurrent, collectively called attached gills. Finer distinctions are often made to distinguish the types of attached gills: adnate gills, which adjoin squarely to the stalk; notched gills, which are notched where they join the top of the stalk; adnexed gills, which curve upward to meet the stalk, and so on. These distinctions between attached gills are sometimes difficult to interpret, since gill attachment may change as the mushroom matures, or with different environmental conditions.

Fig.: 1 Fruiting body of mushroom (Source: https://www.google.com.np/)

1.1.3 Habitat and Ecology Mushrooms generally grow at the places where there is sufficient moisture and favourable temperature. They appear in places where the habitats are undisturbed by man in wild and afforested reserved areas in nature. They appear in moist, open, shady places on soil, burnt ground and grassy lands. Generally the growth of thallus is controlled by different environmental and ecological factors where they retain, the moisture and nutrition necessary for growth, fructification and reproduction. Although mushrooms constitute different mode of habitat but in general it can be described in three basic forms:

Saprophyte eg. Agaricus, Boletus, Coprinus etc. Parasitic eg. Armillaria, Polyporus, Fomes etc. Symbiont or Mycorrhizae eg. Amanita, Russula etc.

The microclimate conditions are directly related to topography and altitude which govern the pattern of growth and distribution. Thus, the presence of a fungus in a particular area depends upon the topography (temperature and rainfall) and particularly the vegetation. Temperature is the most important factor governing distribution (Bakshi, 1971) Temperature is the basic factor that changes the climate although for most mushrooms species, the temperature of the substrate is more important than the air temperature. Thus sun and heat play an important role in the fructification of the higher fungi. In temperate climate the highest production of mushrooms occur in late summer and early autumn, when the atmospheric precipitation lowers the temperature and raises the humidity level of the ground (Pacioni, 1985).

During spring and summer seasons in the tropical belt, the environment is dry and the temperature exceeds 25oC. It favours the growth of phytopathogenic species rather than other fungi. The saprophytic and mycorrhizal fungi grow only after the shower in rainy season. In the subtropical and temperate belts the temperature between 15oC and 25oC favours the growth of different mycotaxa (Adhikari, 2000a).

1.1.4 Mushroom Poisoning

Mushroom poisoning is caused by the consumption of cooked or uncooked fruiting body of mushroom or toadstool. Most of the cases occur when the toxic species

confused with the edible species. All human are susceptible to mushroom poisoning. The poisonous species are ubiquitous and geographical restriction on poisoning that may occur in one location do not exist. Individual species of poisonous mushroom are characterised by individual variation in toxin content based on genetics, geographic mushrooms consumed and the dose of toxin delivered. Normally, non-lethal toxin seriously affect the children than the adults and are more likely to suffer very serious consequences from ingestion of relatively smaller doses. Very old, very young and debilitated person of both the sexes are more likely to become seriously ill from all type of mushroom poisoning even from those types, which are generally considered to be mild (www.gmushrooms.com). More than 95% of mushroom poisoning incidences around the world occur due to misidentification (Thomas 2008). Even though poisonous mushrooms represent less than 1% of the world‟s known mushrooms, we cannot ignore the existence of the relatively few dangerous and sometimes fatal species (Chang, 1999). Death of several historical figures like Roman Emperor Claudius, Pope Clement VII, Tsaritsa Natalia Naryshkina, Holy Roman Emperor Charles VI and Siddharth Gautama are considered to be associated with mushroom poisoning (Stamets 2000, Marmion &Wiedemann 2002).

Mushroom poisoning are generally acute and manifested by variety of symptoms and prognoses depending on the amount and species consumed. Because the chemistry of many mushroom toxins is still unknown and positive identification of the mushroom is often difficult or impossible. Mushroom poisoning is generally categorized by their physiological effects (Svreck, 1975). The characteristic pathologic finding in fatalities from amatoxin containing mushroom poisoning is acute massive necrosis of liver parenchyma, hemolysis and renal failure are common accompaniments with occasional pancreatitis (Patowary, 2010).

Modern phytochemical screening of mushroom revealed the presence of alkaloids, cardiac glycosides, saponins, flavonoids, terpenes, steroids, tannins and phenols causes toxicity to human and other animals (Unekwu et al., 2014). Toxicity may be for short or long duration and prolonged activity.

1.1.5 Nutritional and Medicinal value

Mushrooms are low in calories, high in fiber, and contain many important vitamins and minerals. Some also have medicinal properties such as complex carbohydrates

that strengthen the immune system Mushrooms have been recognized as delicious food of good quality protein. These are rich in vitamins particularly in vitamin C and vitamin B-complex and minerals. These are the food of low calorie with little fat, where sugar content is very less, starch and cholesterol are absent and ergosterol is present.

Carbohydrates

The carbohydrates content of mushrooms represents the bulk of fruiting bodies accounting for 50 to 65 % on dry weight basis. Free sugars amounts to about 11%. Florezak et al. (2004) reported the Coprinus atramentarius Contains 24% of carbohydrates on dry weight basis. The mannitol,also called as mushroom sugar constitutes about 80% of the total free sugars, hence it is dominant (Tseng and Mau, 1999; Wannet et al., 2000). Carbohydrates of Agaricus bisporus were reported by Crisan and Sands (1978).

Proteins

Mushrooms have a good nutritional value especially protein. Purkayastha and Chandra (1976) found 14 to 27% crude protein on dry weight basis in Agaricus bisporus, Lentinus subnudus, Calocybe indica and Volvariella volvaceae but protein content of fresh mushroom is 3.7% as stated by FAO publication (1978). They have high percentage of all amino acids. Protein content of mushrooms depends on the composition of the substratum, size of pileus, harvest time and species of mushrooms (Bano & Rajarathnam, 1982). The protein content of the mushroom is almost equal to that of the corn and milk but more than either potato or cabbage. They are still inferior in protein to such standard protein sources such as meat, fish, egg and cheese but their protein content is twice high as that of vegetables with exception of peas and other legumes (Sohi & Sharma, 1997). Clavaria coralloides and Boletus loyus are best protein sources but are deficient in Methionine and cysteine (Schmeda et al., 1997). On dry matter basis, the protein content of varies between 19/100 and 39/100g (Weaver et al., 1977; Breene, 1990).

Fats

The fat content is very low in mushrooms as compared to carbohydrates and proteins. The fats present in mushroom fruiting bodies are dominated by unsaturated fatty

acids. The crude fat content of mushrooms is 2-8% of the dry weight but it can vary from less than 1% to as high as 15-20 % (Crisan & Sands, 1978). Giri and Rana (2008) reported the lowest crude fat constituent was found in Amanita hemibapha (0.39%) and the highest value was observed in Ramaria botrytis (28.32%). Singer (1961) determined the fat content of some mushrooms as 2.04% in Suillus granulatus, 3.66% in Suillus luteus and 2.32% in Agaricus compestris. The total fat content in Agaricus bisporous was reported source of fats and minerals (Jiskani, 2001). Vitamins

Mushrooms are excellent sources of many B vitamins such as thiamine (B1), Riboflavin (B2), nicotinic acid and pantothenic acid. Vegetables are poor source of vitamin B12. This deficient can be met by consuming only 3 g of mushroom. Mushrooms also contain vitamin C (Ascorbic acid) and Vitamin K. Vitamin A, D and E are found to be present only in low amounts (Sohi & Sharma, 1997).

Mineral constituents

Major mineral constituents in mushrooms are K, P, Na, Ca, Mg and elements like Cu, Zn, Fe, Mo, Cd form minor constituents (Bano et al.,1981, Chang,1982). K, P, Na and Mg constitute about 56 to 70% of total ash content of the mushrooms (Li & Chang, 1982). The mineral proportions vary according to the species, age and the diameter of the fruiting body. It also depends upon the type of the substratum (Demirbas, 2001). The mineral content of wild edible mushrooms has been found higher than cultivated ones (Aleror, 1995; Mattila et al., 2001; Rudawska & Leski, 2005).

Medicinal importance

Mushrooms are famous for excellent health food enriched by good quality protein and multitude of beneficial vitamins and minerals. Mushrooms are a rich source of natural antibiotics. The glucans found in the cell wall are well known for their immunomodulatory properties, and the secondary metabolites have been found to be active against bacteria (Kupra et al., 1979) and viruses (Suzuki et al., 1990). Exudates from mushroom mycelia are active against protozoans such as the malaria parasite Plasmodium falciparum (Isaka et al., 2001). They have been used in medicine since the Neolithic and Paleolithic eras (Samorini, 2001). Mushrooms have been used in health care for treating simple and age old common disease like skin diseases to

present day complex nd pandemic disease like AIDS. They are reputed to possess anti-allergic, anticholesterol, anti-tumor and anti-cancer properties (Jiskani, 2001). Ganoderma lucidum are known to lower blood pressure a (Kabir et al., 1988). Lentinus edodus has been used to enhance vigour, sexuality, energy and as an anti aging agent (Gareth, 1990). Mushrooms medicines are without side effect (Sagakami et al., 1991). Cordyceps has been used to induce restful sleep, acts as anticancer, anti aging and anti asthma agents besides proved effective for memory improvement and as sexual rejuvenator (Sharma, 2008). The traditional uses of mushrooms as medicine are certainly limited. In the far western region of the country (the districts of Bajhang and Baitadi) it was found that the spores of many species of Lycoperdon have been used to heal the wounds,Ganoderma lucidum and Pycnoporus cinnabarinus are generally utilized to heal cuts and wounds. The sporocarps are cut and rubbed in morter with drops of water. The paste obtained is applied against the scratched surfaces. The exact of the fungus is also used against dysentery (Adhikari, 1988ab; Bhandary, 1991).

1.1.6 Ethnomycological aspect

Ethnomycology is the investigation and study about the utilization of mushrooms by different ethnic groups. The ethnomycological aspect is not yet studied in detailed in Nepal. The collection and consumption of mushrooms from wild is common in Nepal. While observing critically the origin and distribution of some groups of ethnic casts, they are found to localize in particular zone, for example the Sherpa and Bhote live in high altitude of Himalayas where it is very cold and there is snow in winter. The middle Himalayan dwellers are Tamang, Limbu, Gurung and Rai. On the contrary, in the Terai plain, the Tharu and Danuwar live in contact with the dry forest of tropical zone. These ethnic groups are the traditional collectors. Their knowledge on mushrooms is quite different (Adhikari, 2000a).

There is a general structure used in the language to distinguish the mushrooms in Nepal. In practical all types of language chyau (Nepali), bammhukan (Newari), shyamu (Tamang), shamu (Sherpa), jhyabo (Gurung), mugan (Magar), pat (Limbu) and chhani (Tharu) (Adhikari, 1976) are being used.

1.2 Objectives, Justification and Limitation

1.2.1 Objectives The main objective of the study was to study the mushrooms diversity of the species collected from Amrite community forest and to analyse the nutrient content in cultivated edible (Pleurotus ostreatus) and wild edible ( Scleroderma cepa) species. The specific objectives are

 To collect different types of mushrooms found in the Amrite community forest.  To examine the traditional mycological knowledge.  To explore edible and poisonous mushrooms.  To analysis the protein and different minerals content in cultivated edible and wild edible species of mushroom.

1.2.2 Justification Nepal has expressed its commitment to develop a national strategy for conservation and sustainable use of biological resources. Therefore it is necessary to have detailed information and knowledge about its natural resources and potentiality. Very few study related to Nepalese mycoflora in comparison with higher plants have been done (Adhikari, 2000a). Amrite community forest is a potential place for the exploration of diversity of wild mushrooms. Therefore the present study will be helpful for the knowledge of the mushrooms in Kapilwsastu district and ethnomycological concept of this area.

1.2.3 Limitations  The study is done for the partial fulfillment of M.Sc. Degree.  All the collected specimens could not be preserved due to their poor stage  All the specimens collected could not be identified up to species level due to immature and over matured stage of specimens.  The information collected is based on field investigation done in June to September 2016 AD in the study area.

CHAPTER TWO

LITERATURE REVIEW

J.D Hooker (1848-1854) explored east Nepal in a botanical survey. The result of his gatherings was published by Berkeley ( 1854 a, b, c, d). He reported 44 higher fungi in “Indian fungi” in Hooker‟s Journal of Botany. The papers included 18 new species for Nepal viz. Irpex zonatus Lentinus nepalensis, L. inquinans, Lycoperdon elongatum, L .emodense, Polyporus cereus , P . elatinus, P . flavidus, P. florideus, P. nepalensis, P. pictilis, Radulum spongiosum, Scleroderma nitidum, Sphaeria nepalensis, Stereum endocrocinum, Trametes tephroleuca, T. versatilis and Xyalaria fistuca.

Thind (1961) included three species in “The Clavariaceae of India‖ Which were reported earlier by Balfour-Browne (1955).

Bhatt (1966) enumerated 118 species of fungi from different parts of Nepal which included Myxomycota-1, Mastigomycotina and Zygomycotina-8, Ascomycotina-8, Basidomycotina-27, Basidiomycotina-33 and Deuteromycotina-51.

Singh (1966) reported 18 wild edible species of mushroom sold at Kathmandu market in bamboo packages. Singh (1968) also reported 4 species of Hymenomycetes from Kathmandu valley.

Poelt (1965) collected 55 species of myxomycetes from Khumbu district and its adjoining area. Two species viz. Arcyria nepalensis and Lamproderma nigrisplendidum were newly reported to science.

Singh and Adhikari (1977) described four genera and five species of fleshy fungi collected from Manichaur, Kathmandu valley. Five species viz. Trichoglossum velutipes, Dacromyces palmatus, Clavaria sp., Clavulinopsis fusiformis and Clavulionopsis sp. were new to Nepal.

Mushrooms have good nutritional value particularly as a source of protein that can enrich human diets especially in some developing countries where animal protein may not be available and are expensive. The protein content of fresh mushroom is 3.7% (FAO, 1978).

Bhandary (1984) prepared a checklist of edible and poisonous mushrooms along with their local names.

Hjortstam and Ryvarden (1984) published the occurrence of 60 genera and 95 species of Basidiomycetes (Aphyllophorales), from Pokhara and Annapurna region. The new species described were Peniophora bicornis, Grammothele bambusicola, Innotus hemisetulus, Phlebia albo – fibillosa and Phellinus acontextus.

Cotter and Bhandary (1985) reported the occurrence of Clavimalum indium (Clavicipitaceae) on Arundinaria sp.

Adhikari (1990a) provided a brief review on history of mycological explorations carried on by the investigators till 1990. The paper recorded about 428 genera and 1200 species. Adhikari (1990b) reported 11 species of the genus Russula collected during a mycological expedition to east Nepal. Among collected species these speies were new to Nepal viz., R. luteotacta, R. nitida, R. olvacea, R. pectinata, R. pseudodelica, and R. subnigricans.

Adhikari and Durrieu (1996) report the consumption of 57 fungi from Khumbu region.

Zang and Kinjo (1998) gave an account of 33 species of the genus Cordyceps collected from the alpine areas of China and Nepal. Among these Cordyceps nepalensis was described as new to science gathered from Kanchanjunga (4300m), Kathmandu valley market.

The edible and medicinal mushrooms can be used on human welfare in the 21st century (Chang, 1999).

Adhikari (2000a) reported 776 species of mushroom belonging to 77 families and 213 genera.

Adhikari (2000b) reported nine genera of Ascomycotina and twenty-eight genera of Basidiomycotina from Maipokhari, East Nepal which were new to that area.

Adhikari (2001) reported wild mushrooms species from Kathmandu valley viz. Hypomyces sp., Leccinum rugosiceps, Pleurotus cornucopiae, Polyporellus varius, Ramaria aurea, R. flava, R. Formosa, Sarcodon laevigatus and Suillus bovines.

Pandey and Budhathoki (2003) reported Rhizinia undulate a wild inedible mushroom from the coniferous Pinus dominant forests of Champadevi, Kirtipur, Kathmandu.

Pandey (2004) analyzed 25 wild mushrooms species from Kathmandu valley for their protein constituent. Chemical study revealed that the higher amount of the total amino acid was detected on Coprinus comatus (13.80mg/ml) followed by Amanita caesarea (13.67mg/ml) and Agaricus bisporous (13.39mg/ml).

Adhikari et al. (2005), reported 18 edible species of mushrooms from Lumle (kaski) and Kathmandu Valley out of 24 species.

Devkota et al. (2005) recorded three Clavariales – Aphelaria tuberso, Clavaria fumosa and Lentaria mucida from Dakshinakali – Kathmandu, Godavary – Lalitpur and Lumle- Kaski respectively.

Giri and Rana (2007) collected 69 species of mushroom from the Sagarmatha National Park and its vicinity which were recorded first time from that area.

Pandey and Budhathoki (2007) determined the protein of 35 species of mushroom through Bradford‟s method and the highest amount of protein (1.576mg/ml) was found in Cantharellus subscibarius and least (0.131 mg/ml) was found in Cordycep sinensis.

Christensen et al. (2008) confirmed 228 species of edible mushrooms in Nepal. They reported Twenty-one of the species commonly used in Nepal are species that are commonly used all over the world.

Devakota (2008), collected 44 macrofungi belonging to 25 families and 37 genera were collected from Raha and Majphal VDCs of Dolpa district. Among the collected species 22 have found with their culinary values and 5 with medicinal values.

Giri and Rana (2008) identified 29 ethnomycologically important mushrooms with the help of Sherpa community residing within Sagarmatha National Park area. Among them 26 species of mushrooms were used for edible, 2 species for medicinal and one for decorative purpose. Chemical analysis of their finding reported 62.63% for Chroogomphus tomentosus, highest crude fat content for Ramaria botrytis (28.32%),

Tylopilus eximus had highest moisture content (19.84 %.), calcium contentent was highest in Paxillus involutus (944 mg/100 gm).

Aryal (2009) reported mushroom poisoning in Nepal and its mitigation.

Jha et al. (2011) studied 11 ethnomedical mushrooms viz., Coprinus comatus, Daldinia concentrica, Fistulina hepatica, Ganoderma appalantum, G. lucidum, G. tsugae, Grifola frondosa, Lepiota cristata, Lycoperdon pyriforme and Pycnoporus cinnabarinus, Trametes versicolorbelonging to 7 families of Nagarjun and Phulchowki of Kathmandu valley and reported that they are used for curing cancer, antitumor, pneumonia, wounds, stops bleeding, constipation, itchiness, blood pressure and diabetes.

Aryal et al. (2012) collected 21 species of macrofungi from Pipaldana Community forest, Saljhandi of Rupandehi district of Western Nepal. The recorded species were categorized statistically on the basis of order and species frequency of occurrence. The studied revealed that order Agaricales had maximum frequency of 33.33% (7 species) followed by order Polyporales 28.57% (6 species).

Jha and Tripathi (2012) studied the macrofungi of Shivapuri National Park of Kathmandu and collected 50 species. Out of these only 22 species belonging to 14 families were identified.

Baral et al. (2015) reported 84 species of macrofungi from the community managed sal (Shorea robusta) forest of Dhading district of central Nepal.

Aryal and Budhathoki (2016) studied recorded 241 species of Ascomycetes and Basidiomycetes belonging to 19 orders, 45 families and 95 genera from 27 sites of Terai Shiwalik and MIdhill region of the country between latitude of 26°44ʹ08ʺ N and 29°06ʹ32ʺ N and longitude of 80°18ʹ02ʺ E and 88°08ʹ27ʺ E.

CHAPTER THREE

MATERIALS AND METHOD

3.1 Study Area

3.1.1 Physiography

The study was conducted in Amrite community forest and its vicinity that lies in Kapilwastu district in the western development region south of the chure hills of Argakhanchi district. Amrite community forest lies 27 41‟ N and 83 06‟38" E to 27044'46.5" N and 83005'48" E. It lies at an elevation of 106 to 140 m from mean sea level in the Kapilwastu district. It occupies nearly about 198.7 hectare of land in Banganga municipality ward number 17. The area is characterized by its low elevation, experiencing tropical monsoon climate of hot, rainy summer and cool dry winter (DNPWC & IUCN 2003).

Amrite community forest was established in 2066 B.S. which is conserved by more than 1200 people that includes 261 residence residing towards its southern vicinity. It is surrounded by Amrite Khola towards East, Dhire Khola towards west and south and way to Amrite cattle pasture land towards North. The forest is tropical forest which consist of Shorea robusta, Adina cordifolia, Acacia catechu, Bombax ceiba, Aegle marmelos, Anthocephalus chinensis, Terminalia alata, Terminalia chebula, Terminalia bellarica, Acorus calamus, Syzygium cuminis, Cassia fistula, etc. The forest occupies 180 years old mature trees mostly of Shorea robusta. During monsoon season, people near its vicinity go to the forest for the collection of edible mushroom which they sell in the local market at the rate of 300 to 600 rupees per kg. The mostly edible mushrooms found in this forest are Amanita hemibapha, Cantharellus spp. and Scleroderma spp.

The soil of this region is mostly gravel and pebbles and hence is highly porous. Water of the mountains, streams disappears here to reissue again in the form of springs in the Terai. Coarse gravel and boulders mixed with ferruginous sand and clay make the soil of this region. The soil of the inner forest is rich in humus due to the high deposition of litter which favours the growth of mushroom under dense canopy of large sal forest.

Fig. 2: Map of Kapilwastu district showing Banganga municipality

Fig. 3: Map of Amrite community forest, Banganga-17, Kapilwastu

3.1.2 Climate

The climate of the study site has a humid, subtropical climate. Its average annual temperatures range from 26°C -16°C with a maximum of 43°C in the summer to a minimum of 4.5°C in the winter. Twenty five years of temperature record in Kapilwastu shows that there is increasing trend of temperature with 0.0216°C/year (Bhandari, 2013). Its average annual rainfall is 1,850 mm, about 80% of which falls during the monsoon season, from mid-June to mid-September.

200 800 760 180 720 680 160 640 600 140 560

520

C) C) 120 480 0 440 100 400

360 Rainfall Rainfall (mm)

Temperature( 80 320 280 60 240 200 40 160 120 20 80 40 0 0 Jan Feb Mar Apr May June July Aug Sep Oct Nov Dec Months

Max. Temp. Min. Temp. Rainfall

Fig. 4 : Ombrothermic representation of climate in the study area (Kapilwastu) from 2013-2016. (Source: Metrological department, Bhairahawa)

3.2 Materials and equipments

The following materials and equipments are necessary for the collection of mushroom (Adhikari, 1991, Brundrett, et al., 1996).

Digital camera, Zipper bags, GPS, Pocket knife, Storage bags, Stationaries: Brush for cleaning specimens, envelops for storing dried specimens, grey color board as background for photographs maps, pen and pencil reference book (field-guide type) Ruler for measuring mushroom Small and large paper bags to keep the collection from the collected area, Small notebook for recording data, white and black paper for spore prints and first aids materials. 3.3 Chemicals:

95% ethyl alcohol was used for the liquid preservation for the samples.

The field work was conducted five times (June to September 2016) in the “Amrite Community Forest” of the Banganga Municipality, Kapilwastu. While conducting field trips local people were accompanied. Moving dense forest in the month of the hot rainy season there is a risk of wild animals as well as snakes was challenging task. Indigenous knowledge survey was conducted. The Participatory Rural Appraisal (PRA) technique was adopted with the local people aimed at getting information largely on nutritional aspects. Data were obtained using combined semi-structured questionnaire, participatory discussions and field observations. Data were analysed using SPSS and Excel softwares.

The mushrooms were photographed in their natural habitat before they were collected. The broken, rotten species were discarded. The basidiocarps were picked up by digging them out carefully with the help of sharp knife. Attempts were made to collect all the developmental stages of basidiocarps to have idea of all morphological characters, keeping in mind „Few good specimens are better than several ones‟ (Adhikari, 1991).

Specific collection numbers were given for each species. Either each of same species or different species collected from the field were cleaned with the help of brush. Paper bags were used for the collection of specimens in the field.

3.4 Preservation of Mushrooms

The mushroom specimens were preserved by two ways

3.4.1 Dry preservation

The specimens collected were dried immediately in order to prevent from rotting. Sun drying was suitable for dry mushrooms. Flesh specimens could not be dried only in sunlight. Wire tray with mushrooms specimens were placed over the hearth in near about 40oc to dry mushrooms. After well drying, the specimens were packed in cellulose paper bags with some naphthalene bulbs and shield in auto lock plastic bags to prevent from decay and insects (Atri & Saini, 2000).

3.4.2 Liquid preservation

Particularly fleshy mushrooms are preserved in the liquid. For the preservation 95% alcohol is used of the preservation of mushroom specimens (Ainsworth, 1971).

3.5 Microscopic study

From the microscopic studies, the fleshy preserved specimens were taken. The slides of the gills or pore were made with the help of forceps. During slide preparation cotton blue and lactophenol were used. Then after the slides were observed with the help of microscope and shape and size were observed. The sizes of the spores were calculated with the help of the ocular micrometer.

3.6 Spore print

For taking spore prints, stipe of the fruit body was cut and the cap was set on a piece of white paper(for white spored fungi black paper was used and for other color spored fungi white paper was used) turning the gills downwards. A drop of water was mounted on the cap in order to minimize dry out of tissues. The material was then placed on a container and incubated for sometimes (2-24hrs) depending on the nature of the fruit body. Finally, the spore prints were photographed (Kuo, 2006).

3.7 Identification of mushrooms

The specimens were studied at Central Department of Botany, TU Kirtipur. The specimens that were collected and preserved were identified with the help of relevant literatures (Pacioni, 1985; Singer, 1986; Adhikari, 2000a).

3.8 Nutrient Analysis

Nutrients content (Iron, Manganese, Calcium, Magnesium, Sodium and Potassium) in cultivated edible (Pleurotus ostreatus) and wild edible species ( Scleroderma cepa) were determined by Atomic absorption spectrophotometer method mentioned by American Public Health Association (APHA) Method 3111B 21st edition. Phosphate (PO4-) content was determined by APHA method 4500 -PE. APHA 21st edition. Protein content (%) was determined by titration method after extraction of nitrogen content mentioned by Association of Official Analytical Chemists (AOAC) 14th edition, 1984.

Total Phosphate

Phosphate occurs in orthophosphate, condensed phosphate and organic phosphate. Orthophosphate responds to the colorimetric determination without treatment and hence it is called reactive phosphate. Polyphosphate as well as organically bound phosphate gives no or very less response towards the colorimetric determination and hence it is required to convert such organically bound and poly phosphate into orthophosphate prior to determination of total phosphate. After conversion of the all forms of phosphates into orthophosphate, the total phosphate can be determined by ascorbic acid method.

Reagents

a) Sulphuric acid (5N): Dilute 70 ml conc. Sulphuric acid to 500 ml with water. b) Potassium antimony tartarate solution: Dissolve 1.3715gm of potassium antimony tartarate [K (SbO)

C4H4O6.H2O] in 400ml distilled water in 500ml volumetric flask and dilute to volume with water. c) Ammonium molybdate solution:

Dissolve 20gm of ammonium molybdate [(NH4)6 MO7O24. 4H2O] in 500ml distilled water. d) Ascorbic acid solution (0.01M):

Dissolve 1.76gm of ascorbic acid [C6H8O6] in 100ml distilled water. e) Combined reagent:

Add 50ml of 5N H2SO4, 5ml of potassium antimony tartarate, 15ml of ammonium molybdate and 30ml of ascorbic acid solution respectively and let stand for few minutes. (Note: It should be prepared at the time of use.)

f) Stock phosphate standard (50ppm, PO4 - P): Dissolve 0.2195gm of anhydrous potassium dihydrogen

orthophosphate [KH2PO4] in distilled water and adjust the volume in

1000ml volumetric flask. This solution contains 50.0ug PO4-P/ml.

From this stock standard solution prepare 10ppm PO4 - P standard solution by diluting 20 cc of it to

Procedure

1. Pipette adequate amount (50 - 100ml) of sample in a 250ml beaker. 2. Add 5ml of Conc. nitric acid followed by 1ml conc. sulphuric acid. 3. Digest over hot plate up to white fumes commences. 4. Cool & wash the interior of the beaker with small volume of distilled water. Neutralize the solution to phenolphthalein indicator (pink at end point with sodium hydroxide solution) and remove the pink colour with 5N sulphuric acid solution. Then allowed to cool. 5. Adjust the volume in 100ml volumetric flask with distilled water and let it stand for the settling of precipitate if formed. 6. Prepare blank with using distilled water as sample proceeded as sample treatment. 7. Pipette 50ml of blank and treated sample solution in 100ml Erlenmeyer flask separately. 8. Similarly take 1.0, 2.0 and 3.0 ml of working standard solution in 100 ml volumetric flasks and adjust volume to 50 ml with water. 9. Then add 8ml of combined reagent.

10. Let it stand for 10 - 15min for complete colour development and read the absorbance at 880nm. The concentration of phosphate was calculated.

Calculation

Total phosphate (PO4), mg/l = (x × f × V × y) / v × 50. Where, x = Concentration (g) from graph directly. V = Volume prepared after digestion. f = Multiplying factor, 95 / 31 y = Dilution factor v = Volume taken for analysis Sample Pre - Treatment:

It is necessary to treat the sample properly prior to estimation of the trace metals levels on it. The pre-treatment may be either the concentration of the sample or the complete acid digestion to convert undissolved metals to the solution. The concentration or the reduction in volume can be carried out by the slow evaporation of the sample over hot plate in an acidic condition.

Procedure of the Concentration

* Take proper amount (100-150 ml) of the sample in a 250 ml capacity acid washed beaker. * Add 1.0 ml of concentrated nitric acid and evaporate slowly over the hot plate. * After adequate reduction in volume, cool it and wash the interior of the beaker with small amount of water. * Transfer the content in a volumetric flask (10 ml) and wash the beaker with a small portion of the water. * Transfer each washing to the same volumetric flask and finally adjust volume up to mark with water.

Procedure of the Sample Digestion * Take appropriate volume (50-100 ml) of the well-mixed sample into a clean beaker. * Add 5.0 ml concentrated nitric acid and a few boiling chips on it.

* Heat over hot plate carry out slow evaporation to the lowest possible volume. * If necessary, continue heating by adding conc. Nitric acid till it gives light colored clear solution. * After digestion, cool the beaker and wash down the interior walls with small amount of water and then filter if necessary. * Transfer the filtrate into the volumetric flask and after cooling down to room temperature adjusts the required volume with water.

Determination of the Metal Concentration After the pre treatment of the sample, determination of the trace metal level is carried out by direct air-acetylene flame method. Both emission and absorption mode can be used depending upon the element. Also Manufacturer‟s specification and Cookbook of the Atomic Absorption Spectrophotometeter (AAS).

1. Analysis of trace Elements (Sodium and Potassium) was conducted by Flame Emission Method in air acetylene flame (3111 B. APHA, 21ST EDITION)

2. Analysis of Heavy and Nutrient Metals was conducted by Absorption Method (Iron, Manganese and Magnesium) by Acetylene - Air Flame (3111 B, APHA, 21ST EDITION)

3. Analysis of Nutrient Metals (Calcium) was conducted by Absorption Method (Iron, Manganese and Magnesium) by Nitrous Oxide - Air Flame (3111 D, APHA, 21ST EDITION)

4. Analysis of Protein was conducted by Kjeldahl digestion method after extraction.

3111 B. Direct Air–Acetylene Flame Method

1. General Discussion This method is applicable to the determination of antimony, bismuth, cadmium, calcium, cesium, chromium, cobalt, copper, gold, iridium, iron, lead, lithium,

magnesium, manganese, nickel, palladium, platinum, potassium, rhodium, ruthenium, silver, sodium, strontium, thallium, tin, and zinc.

2. Apparatus Atomic absorption spectrometer and associated equipment: See Section 3111A.6. Use burner head recommended by the manufacturer.

3. Reagents a. Air cleaned and dried through a suitable filter to remove oil, water, and other foreign substances. The source may be a compressor or commercially bottled gas. b. Acetylene, standard commercial grade. Acetone, which always is present in acetylene cylinders, can be prevented from entering and damaging the burner head by replacing a cylinder when its pressure has fallen to 689 kPa (100 psi) acetylene. c. Metal–free water: Use metal–free water for preparing all reagents and calibration standards and as dilution water. Prepare metal–free water by deionizing tap water and/or by using one of the following processes, depending on the metal concentration in the sample: single distillation, redistillation, or sub–boiling. Always check deionized or distilled water to determine whether the element of interest is present in trace amounts.

d. Calcium solution: Dissolve 630 mg calcium carbonate, CaCO3, in 50 mL of 1 + 5 HCl. If necessary, boil gently to obtain complete solution. Cool and dilute to 1000 mL with water. e. Hydrochloric acid, HCl, 1%, 10%, 20%, 1 + 5, 1 + 1, and conc.

f. Lanthanum solution: Dissolve 58.65 g lanthanum oxide, La2O3, in 250 mL conc HCl. Add acid slowly until the material is dissolved and dilute to 1000 mL with water. g. Hydrogen peroxide, 30%.

h. Nitric acid, HNO3, 2%, 1 + 1 and conc.

i. Aqua regia: Add 3 volumes conc HCl to 1 volume conc HNO3. j. Standard metal solutions: Prepare a series of standard metal solutions in the optimum concentration range by appropriate dilution of the following stock metal solutions with water containing 1.5 mL conc HNO3/L. Thoroughly dry reagents before use. In general, use reagents of the highest purity. For hydrates, use fresh reagents.

1) Calcium: Suspend 0.2497 g CaCO3 (dried at 180° for 1 h before weighing) in

water and dissolve cautiously with a minimum amount of 1 + 1 HNO3. Add

10.0 mL conc HNO3 and dilute to 1000 mL with water; 1.00 mL = 100 µg Ca. 2) Iron: Dissolve 0.100 g iron wire in a mixture of 10 mL 1 + 1 HCl and 3 mL

conc HNO3. Add 5 mL conc HNO3 and dilute to 1000 mL with water: 1.00 mL = 100 µg Fe.

3) Magnesium: Dissolve 0.1658 g MgO in a minimum amount of 1 + 1 HNO3.

Add 10.0 mL conc HNO3 and dilute to 1000 mL with water; 1.00 mL = 100 µg Mg. 4) Manganese: Dissolve 0.1000 g manganese metal in 10 mL conc HCl mixed

with 1 mL conc HNO3. Dilute to 1000 mL with water; 1.00 mL = 100 µg Mn. 5) Potassium: Dissolve 0.1907 g potassium chloride. KCl. (dried at 110°C) in water and make up to 1000 mL: 1.00 mL = 100 µg K. 6) Sodium: Dissolve 0.2542 g sodium chloride, NaCl, dried at 140°C, in water,

add 10 mL cone HNO3 and make up to 1000 mL: 1.00 mL =100 µg Na.

4. Procedure a. Sample preparation: Required sample preparation depends on need to measure dissolved metals only or total metals. If dissolved metals are to be determined, see Section 3030B for sample preparation. If total or acid-extractable metals are to be determined, see Sections 3030C through K. For all samples, make certain that the concentrations of acid and matrix modifiers are the same in both samples and standards. When determining Ca or Mg, dilute and mix 100 mL sample or standard with 10 mL lanthanum solution (3f) before atomization. When determining Fe or Mn, mix 100 mL with 25 mL of Ca solution (3d) before aspirating. When determining Cr, mix

1 mL 30% H2O2 with each 100 mL before aspirating. Alternatively use proportionally smaller volumes. b. Instrument operation: Because of differences between makes and models of atomic absorption spectrometers, it is not possible to formulate instructions applicable to every instrument. See manufacturer‟s operating manual. In general proceed according to the following: Install a hollow-cathode lamp for the desired

Metal in the instrument and roughly set the wavelength dial according to Table 3111:I. Set slit width according to manufacturer‟s suggested setting for the element being measured. Turn on instrument, apply to the hollow-cathode lamp the current suggested by the manufacturer, and let instrument warm up until energy source stabilizes, generally about 10 to 20 min. Readjust current as necessary after warm up. Optimize wavelength by adjusting wavelength dial until optimum energy gain is obtained. Align lamp in accordance with manufacturer‟s instructions.

Install suitable burner head and adjust burner head position. Turn on air and adjust flow rate to that specified by manufacturer to give maximum sensitivity for the metal being measured. Turn on acetylene, adjust flow rate to value specified, and ignite flame. Let flame stabilize for a few minutes. Aspirate a blank consisting of either deionized water or an acid solution containing the same concentration of acid in standards and samples. Zero the instrument. Aspirate a standard solution and adjust aspiration rate of the nebulizer to obtain maximum sensitivity. Adjust burner both vertically and horizontally to obtain maximum response. Aspirate blank again and rezero the instrument. Aspirate a standard near the middle of the linear range. Record absorbance of this standard when freshly prepared and with a new hollow-cathode lamp. Refer to these data on subsequent determinations of the same element to check consistency of instrument setup and aging of hollow-cathode lamp and standard.

The instrument now is ready to operate. When analyses are finished, extinguish flame by turning off first acetylene and then air. c. Standardization: Select at least three concentrations of each standard metal solution (prepared as in 3j above) to bracket the expected metal concentration of a sample. Aspirate blank and zero the instrument. Then aspirate each standard in turn into flame and record absorbance.

Prepare a calibration curve by plotting on linear graph paper absorbance of standards versus their concentrations. For instruments equipped with direct concentration readout, this step is unnecessary. With some instruments it may be necessary to convert percent absorption to absorbance by using a table generally provided by the manufacturer. Plot calibration curves for Ca and Mg based on original concentration of standards before dilution with lanthanum solution. Plot calibration curves for Fe

and Mn based on original concentration of standards before dilution with Ca solution. Plot calibration curve for Cr based on original concentration of standard before addition of H2O2. d. Analysis of samples: Rinse nebulizer by aspirating water containing 1.5 mL conc

HNO3/L. Atomize blank and zero instrument. Atomize sample and determine its absorbance.

5. Calculations Calculate concentration of each metal ion, in micrograms per liter for trace elements, and in milligrams per liter for more common metals, by referring to the appropriate calibration curve prepared according to 4c. Alternatively, read concentration directly from the instrument readout if the instrument is so equipped. If the sample has been diluted, multiply by the appropriate dilution factor.

3111 C. Extraction/Air-Acetylene Flame Method

1. General Discussion This method is suitable for the determination of low concentrations of cadmium, chromium, cobalt, copper, iron, lead, manganese, nickel, silver, and zinc. The method consists of chelation with ammonium pyrrolidine dithiocarbamate (APDC) and extraction into methyl isobutyl ketone (MIBK) followed by aspiration into an air- acetylene flame.

2. Apparatus a. Atomic absorption spectrometer and associated equipment: See Section 3111A.6. b. Burner head, conventional. Consult manufacturer‟s operating manual for suggested burner head.

3. Reagents a. Air: See 3111B.3a. b. Acetylene: See 3111B.3b. c. Metal-free water: See 3111B. 3c. d. Methyl isobutyl ketone (MIBK), reagent grade. For trace analysis, purify. MIBK by redistillation or by sub-boiling distillation.

e. Ammonium pyrrolidine dithiocarbamate (APDC) solution: Dissolve 4 g APDC in 100 mL water. If necessary, purify APDC with an equal volume of MIBK. Shake 30 s in a separatory funnel, let separate, and withdraw lower portion. Discard MIBK layer.

f. Nitric acid, HNO3, cone, ultrapure. g. Standard metal solutions: See 3111B. 3j.

h. Potassium permanganate solution, KMnO4, 5% aqueous.

i. Sodium sulfate, Na2SO4, anhydrous. j. Water-saturated MIBK: Mix one part purified MIBK with one part water in a separatory funnel. Shake 30 s and let separate. Discard aqueous layer. Save MIBK layer. k. Hydroxylamine hydrochloride solution, 10%.

4. Procedure a. Instrument operation: See Section 3111B.4b. After final adjusting of burner position, aspirate water-saturated MIBK into flame and gradually reduce fuel flow until flame is similar to thatbefore aspiration of solvent. b. Standardization: Select at least three concentrations of standard metal solutions (prepared as in 3111B.3j) to bracket expected sample metal concentration and to be, after extraction, in the optimum concentration range of the instrument. Adjust 100 mL of each standard and 100 mL of a metal-free water blank to pH 3 by adding 1NHNO3 or 1N NaOH. For individual element extraction, use the following pH ranges to obtain optimum extraction efficiency: Transfer each standard solution and blank to individual 200-mL volumetric flasks, add 1 mL APDC solution, and shake to mix. Add 10 mL MIBK and shake vigorously for 30 s. (The maximum volume ratio of sample to MIBK is 40.) Let contents of each flask separate into aqueous and organic layers, then carefully add water (adjusted to the same pH at which the extraction was carried out) down the side of each flask to bring the organic layer into the neck and accessible to the aspirating tube. Aspirate organic extracts directly into the flame (zeroing instrument on a water- saturated MIBK blank) and record absorbance. Prepare a calibration curve by plotting on linear graph paper absorbances of extracted standards against their concentrations before extraction.

c. Analysis of samples: Prepare samples in the same manner as the standards. Rinse atomizer by aspirating water-saturated MIBK. Aspirate organic extracts treated as above directly into the flame and record absorbances. With the above extraction procedure only hexavalent chromium is measured. To determine total chromium oxidize trivalent chromium to hexavalent chromium by bringing sample to a boil and adding sufficient KMnO4 solution dropwise to give a persistent pink color while the solution is boiled for 10 min. Destroy excess

KMnO4 by adding 1 to 2 drops hydroxylamine hydrochloride solution to the boiling solution, allowing 2 min for the reaction to proceed. If pink color persists, add 1 to 2 more drops hydroxylamine hydrochloride solution and wait 2 min. Heat an additional 5 min. Cool, extract with MIBK, and aspirate. During extraction, if an emulsion forms at the water-MIBK interface, add anhydrous

Na2SO4 to obtain a homogeneous organic phase. In that case, also add Na2SO4 to all standards and blanks. To avoid problems associated with instability of extracted metal complexes, determine metals immediately after extraction.

5. Calculations Calculate the concentration of each metal ion in micrograms per liter by referring to the appropriate calibration curve.

3111 D. Direct Nitrous Oxide-Acetylene Flame Method

1. General Discussion This method is applicable to the determination of Iron, Manganese and Magnesium 2. Apparatus a. Atomic absorption spectrometer and associated equipment: See Section 3111A.6. b. Nitrous oxide burner head: Use special burner head as suggested in manufacturer‟s manual. At roughly 20-min intervals of operation it may be necessary to dislodge the carbon crust that forms along the slit surface with a carbon rod or appropriate alternative. c. T-junction valve or other switching valve for rapidly changing from nitrous oxide to air, so that flame can be turned on or off with air as oxidant to prevent flashbacks.

3. Reagents a. Air: See 3111B.3a. b. Acetylene: See 3111B.3b. c. Metal-free water: See 3111B.3c. d. Hydrochloric acid, HCl, 1N, 1+1, and conc.

e. Nitric acid, HNO3, conc.

f. Sulfuric acid, H2SO4, 1%. g. Hydrofluoric acid, HF, 1N. h. Nitrous oxide, commercially available cylinders. Fit nitrous oxide cylinder with a special non freezable regulator or wrap a heating coil around an ordinary regulator to prevent flashback at the burner caused by reduction in nitrous oxide flow through a frozen regulator. (Some atomic absorption instruments have automatic gas control systems that will shut down a nitrous oxideacetylene flame safely in the event of a reduction in nitrous oxide flow rate.) i. Potassium chloride solution: Dissolve 250 g KCl in water and dilute to 1000 mL.

j. Aluminum nitrate solution: Dissolve 139 g Al(NO3)3 9H2O in 150 mL water.

Acidify slightly with conc HNO3 to preclude possible hydrolysis and precipitation. Warm to dissolve completely. Cool and dilute to 200 mL. k. Standard metal solutions: Prepare a series of standard metal solutions in the optimum concentration ranges by appropriate dilution of the following stock

metal solutions with water containing 1.5 mL conc HNO3/L: 1) Aluminum: Dissolve 0.100 g aluminum metal in an acid mixture of 4 mL 1 +

1 HCl and 1 mL conc HNO3 in a beaker. Warm gently to effect solution. Transfer to a 1-L flask, add 10 mL 1 + 1 HCl, and dilute to 1000 mL with water; 1.00 mL = 100 µg Al

2) Barium: Dissolve 0.1516 g BaCl2 (dried at 250° for 2 h), in about 10 mL water with 1 mL 1 + 1 HCl. Add 10.0 mL 1 + 1 HCl and dilute to 1000 mL with water: 1.00 mL = 100 µg Ba.

3) Beryllium: Do not dry. Dissolve 1.966 g BeSO4-4H2O in water, add 10.0 mL

conc HNO3, and dilute to 1000 mL with water; 1.00 mL = 100 µg Be.

4) Molybdenum: Dissolve 0.2043 g (NH4)2MoO4 in water and dilute to 1000 mL; 1.00 mL = 100 µg Mo.

5) Osmium: Obtain standard 0.1M osmium tetroxide solution* and store in glass bottle; 1.00 mL = 19.02 mg Os. Make dilutions daily as needed using 1%

(v/v) H2SO4 CAUTION: OsO4is extremely toxic and highly volatile.

6) Rhenium: Dissolve 0.1554 g potassium perrhenate, KReO4, in 200 mL water.

Dilute to 1000 mL with 1% (v/v) H2SO4: 1.00 mL = 100 µg Re.

7) Silica: Do not dry. Dissolve 0.4730 g Na2SiO3 9H2O in water. Add 10.0 mL

conc HNO3 and dilute to 1000 mL with water. 1.00 mL = 100 µg Si. Store in polyethylene.

8) Thorium: Dissolve 0.238 g thorium nitrate, Th(NO3)4 4H2O in 1000 mL water; 1.00 mL = 100 µg Th.

9) Titanium: Dissolve 0.3960 g pure (99.8 or 99.9%) titanium chloride. TiCl4, † in a mixture of equal volumes of 1N HCl and 1N HF. Make up to 1000 mL with this acid mixture; 1.00 mL = 100 µg Ti.

10) Vanadium: Dissolve 0.2297 g ammonium metavanadate, NH4VO3, in a

minimum amount of conc HNO3. Heat to dissolve. Add 10 mL conc HNO3, and dilute to 1000 mL with water; 1.00 mL = 100 µg V.

4. Procedure a. Sample preparation: See Section 3111B.4a.

b. Instrument operation: See Section 3111B.4b. After adjusting wavelength, install a nitrous oxide burner head. Turn on acetylene (without igniting flame) and adjust flow rate to value specified by manufacturer for a nitrous oxide-acetylene flame. Turn off acetylene. With both air and nitrous oxide supplies turned on, set T- junction valve to nitrous oxide and adjust flow rate according to manufacturer‟s specifications. Turn switching valve to the air position and verify that flow rate is the same. Turn acetylene on and ignite to a bright yellow flame. With a rapid motion, turn switching valve to nitrous oxide. The flame should have a red cone above the burner. If it does not, adjust fuel flow to obtain red cone. After nitrous oxide flame has been ignited, let burner come to thermal equilibrium before beginning analysis.

Atomize water containing 1.5 mL conc HNO3/L and check aspiration rate. Adjust if necessary to a rate between 3 and 5 mL/min. Atomize a standard of the desired metal with a concentration near the midpoint of the optimum concentration range and adjust

burner (both horizontally and vertically) in the light path to obtain maximum response. The instrument now is ready to run standards and samples.

To extinguish flame, turn switching valve from nitrous oxide to air and turn off acetylene. This procedure eliminates the danger of flashback that may occur on direct ignition or shutdown of nitrous oxide and acetylene.

c. Standardization: Select at least three concentrations of standard metal solutions (prepared as in 3k) to bracket the expected metal concentration of a sample. Aspirate each in turn into the flame. Record absorbances. For Al, Ba, and Ti, add 2 mL KCl solution to 100 mL standard before aspiration. For Mo and V add 2 mL

Al(NO3)3 9H2O solution to 100 mL standard before aspiration.

Most modern instruments are equipped with microprocessors and digital readout which permit calibration in direct concentration terms. If instrument is not so equipped, prepare a calibration curve by plotting on linear graph paper absorbance of standards versus concentration. Plot calibration curves for Al, Ba, and Ti based on original concentration of standard before adding KCl solution. Plot calibration curves for Mo and V based on original concentration of standard before adding

Al(NO3)3 solution.

d. Analysis of samples: Rinse atomizer by aspirating water containing 1.5 mL conc HNO3/L and zero instrument. Atomize a sample and determine its absorbance. When determining Al, Ba, and Ti, add 2 mL KCl solution to 100 mL sample before atomization. For Mo and V. add 2 mL Al(NO3)3 9H2O solution to 100 mL sample before atomization.

5. Calculations Calculate concentration of each metal ion in micrograms per liter by referring to the appropriate calibration curve prepared according to 4c. Alternatively, read the concentration directly from the instrument readout if the instrument is so equipped. If sample has been diluted, multiply by the appropriate dilution factor.

Analysis of Proteins

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino- acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. These reactions can have either a favorable or detrimental effect on the overall properties of foods. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods.

Determination of Overall Protein Concentration by Kjeldahl method

The Kjeldahl method was developed in 1883 by a brewer called Johann Kjeldahl. A food is digested with a strong acid so that it releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements. It is usually considered to be the standard method of determining protein concentration. Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications, however, this is only an average value, and each protein has a different conversion factor depending on its amino-acid composition. The Kjeldahl

method can conveniently be divided into three steps: digestion, neutralization and titration.

Principles 1. Digestion The food sample to be analyzed is weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction). Digestion converts any nitrogen in the food (other than that which is in the form of nitrates or nitrites) into ammonia, and other organic matter to C02 and H20. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the + 2- ammonium ion (NH4 ) which binds to the sulfate ion (SO4 ) and thus remains in solution:

N(food) --> (NH4)2SO4 (1)

2. Neutralization

After the digestion has been completed the digestion flask is connected to a recieving flask by a tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide, which converts the ammonium sulfate into ammonia gas:

(NH4)2SO4 + 2 NaOH  2NH3 + 2H2O + Na2SO4 (2)

The ammonia gas that is formed is liberated from the solution and moves out of the digestion flask and into the receiving flask - which contains an excess of boric acid. The low pH of the solution in the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts the boric acid to the borate ion:

+ - NH3 + H3BO3 (boric acid)  NH4 + H2BO3 (borate ion) (3)

3. Titration The nitrogen content is then estimated by titration of the ammonium borate formed with standard sulfuric or hydrochloric acid, using a suitable indicator to determine the end-point of the reaction.

- + H2BO3 + H  H3BO3 (4) The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to the concentration of nitrogen that was in the original food (Equation 3). The following equation can be used to determine the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration:

( ) (5)

Where vs and vb are the titration volumes of the sample and blank, and 14g is the molecular weight of nitrogen N. A blank sample is usually run at the same time as the material being analyzed to take into account any residual nitrogen which may be in the reagents used to carry out the analysis. Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor: %Protein = F× %N. Where, F = Conversion factor (6.25)

CHAPTER FOUR

RESULTS

4.1 Mushroom composition

A total of 38 wild mushroom specimens were collected from the Amrite Community forest of Banganga municipality ward number 16. Out of these 34 species were identified up to generic level belonging to 16 families and 8 orders. On the basis of order, the most dominant order was Agaricales having 16 species (Table 1).

Table 1: List of mushrooms collected from the Amrite Community Forest, Banganga- 17, Kapilwastu.

CN C. Date Host/substrate Lat./Long. Alt. Scientific name Local Order Family Application name (m)

1 2073/3/14 soil 27o41'01.5"/ 114 Macrolapiota procera Gobre Agaricales Agaricaceae Non-edible 83o06ꞌ38" (Scop.) Singer chyau

2 2073/3/14 soil 27o41'11.5"/ 112 Tricholar sp. Agaricales Tricholomataceae Medicinal 83o05ꞌ36"

3 2073/3/14 soil 27o41'12.5"/ 122 Russula spp. Raktey Russulales Russulaceae Edible 83o06ꞌ28"

4 2073/3/14 soil 27o41'21"/ 121 Russula emetica Kali Chyau Russulales Russulaceae Edible 83o06ꞌ48" (Schaeff.) Pers.

5 2073/3/14 soil 27o42'22"/ 136 Pycnoporus Kane Polyporales Polyporaceae Medicinal 83o08ꞌ38" cinnabarinus Chyau (Jacq.) P.Karst.

6 2073/3/14 soil 27o41'25"/ 127 Scleroderma texense Bhutki Bolatales Sclerodermataceae Edible 83o06ꞌ14" Berk.

7 2073/3/14 soil 27o42'17"/ 110 Coprinus Commatus Gobre Agaricales Coprinaceae Unknown 83o06ꞌ36" (O.F.Müll.) Pers.

8 2073/3/14 Soil 27o41'03"/ 122 Amanita chepangiana Salle Agaricales Amanitaceae Edible 83o05ꞌ44" Tulloss & Bhandary Chyau

9 2073/3/14 Soil 27o41'50"/ 112 Buwaldo boletus spp. Dhabre Bolatales Boletaceae Poisonous 83o05ꞌ22" chyau

10 2073/3/14 soil 27o43'19"/ 112 Scleroderma cepa Pers. Bhutki Bolatales Sclerodermataceae Edible 83o05ꞌ07"

11 2073/3/21 soil 27o44'01"/ 114 Amanita sp. Besera Agaricales Amanitaceae Edible 83o05ꞌ20" Cheyu

12 2073/3/21 Soil 27o42'05"/ 118 Russula spp. Ratteuo Russulales Russulaceae Edible 83o05ꞌ20" Chyau

13 2073/3/21 Soil 27o42'44"/ 140 Boletus spp. Bhude Bolatales Boletaceae Medicinal 83o05ꞌ16" Chyau

14 2073/3/21 wood 27o41'01.5"/ 114 Sparissis crispa Cauli Polyporale Sparassidaceae 83o06ꞌ38" (Wolfen) Fr. Chyau

15 2073/3/21 wood 27o42'15"/ 123 Guepinia spathularia Putali Tramellales Dacrymycetacea 83o06ꞌ31" (Schwein.) Fr. Chyau

16 2073/3/21 wood 27o41'11.5"/ 112 Nebularia sp. Peltigerales Pannariaceae 83o05ꞌ36"

17 2073/4/2 dung 27o42'22"/ 132 Coprinus plicalitis Fr. Gobre Agaricales Coprinaceae Inedible 83o05ꞌ06" ex Curtis Chyau

18 2073/4/2 soil 27o42'44"/ 140 Termitomyces Dhamire Agaricales Tricholomataceae 83o05ꞌ16 aurantiacus (R Heim) Chyau R. Heim

19 2073/4/2 soil 27o42'05"/ 118 Lacaria laccata Dudhe Agaricales Hydnangiaceae 83o05ꞌ20" (Scop.) Cooke Chyau

20 2073/4/16 animal dung 27o42'15"/ 123 Coprinus Gobre Agaricales Coprinaceae 83o06ꞌ31" disseminates(Pers.) J.E. chyeu Lange

21 2073/4/16 tree trunk 27o42'05"/ 118 Polyprous durus Kathe Polyporales Polyporaceae Edible 83o05ꞌ20" chyeu

22 2073/4/16 tree trunk 27o42'44"/ 140 Auricularia auricula- Todke Agaricales Auriculariaceae Medicinal 83o05ꞌ16 jude (Bull.) J.Schröt.

23 2073/4/16 wood 27o42'05"/ 118 unidentified Inedible 83o05ꞌ20"

24 2073/4/16 soil 27o41'11.5"/ 112 unidentified 83o05ꞌ36"

25 2073/4/26 Wood 27o42'22"/ 132 Microporous xanthpus Kathe Polyporales Polyporaceae Medicinal 83o05ꞌ06" chyau

26 2073/4/26 wood 27o42'05"/ 118 Daldenia sp. Dalley Polyporales Xylariaceae 83o05ꞌ20"

27 2073/4/26 Soil 27o41'01.5"/ 114 Amanita hemibapha Salle chyau Agaricales Amanitaceae Edible 83o06ꞌ38" (Berk. & Broome) Sacc .

28 2073/4/26 Wood 27o44'02"/ 129 Cantharellus sp. Chamre Cantharella Cantharellacae Edible 83o06ꞌ33" Chyaue les

29 2073/4/26 Soil 27o42'15"/ 123 Amanita sp. Salle Agaricales Amanitaceae Poisonous 83o06ꞌ31"

30 2073/4/26 Rock 27o44'02"/ 129 Unidentified 83o06ꞌ33"

31 2073/4/26 Soil 27o42'15"/ 123 Coprinus sp. Gobre Agaricales Coprinaceae 83o06ꞌ31"

32 2073/4/26 Soil 27o42'44"/ 140 Marasmius perforans Bulaki Agaricales Tricholomataceae 83o05ꞌ16 (Hoffm.) Fr. Chyau

33 2073/5/8 soil 27o44'02"/ 129 Unidentified Kukurmuta Inedible 83o06ꞌ33"

34 2073/5/8 animal dung 27o42'22"/ 138 Coprinus Gobre Agaricales Coprinaceae 83o05ꞌ06" atramentarius (Bull.) Chyau Re

35 2073/5/8 animal dung 27o41'11.5"/ 112 Agaricus sp. Gobre Agaricales Agaricaceae Edible 83o05ꞌ36" chyau

36 2073/5/8 Soil 27o42'22"/ 132 Scleroderma bovista Dalle chyau Boletales Sclerodermataceae Medicinal 83o05ꞌ06" Fr.

37 2073/5/8 Soil 27o41'01.5"/ 114 Cantharellus cibarius Besare Cantharella Cantherallaceae Edible 83o06ꞌ38" Fr. chyau les

38 2073/5/16 Soil 27o44'02"/ 129 Dictyophora indusiata Jali Chyau Phallales Phallaceae Poisonous 83o06ꞌ33" (Vent.) Desv.

The most dominant family was Coprinaceae belonging to 5 species followed by Amanitaceae consisting of 4 species. Russulaceae, Polyporaceae, Tricholomataceae and Sclerodermataceae were represented by 3 species each while Agaricaceae, Boletaceae, Cantheralleceae were represented by 2 species each. Family Auriculariaceae, Dacrymycetaceae, Hydnangiaceae, Pannariaceae, Phallaceae, and Xylariaceae were represented by 1 species each where as four species were unidentified.

Fig. 5: Number of species with respect to families.

The specimens collected like Marasmius, Auricularia, Russula,Aminta, Cantharellus, Coprinus have short life period where as Pleurotus, Scleroderma, Polyporus have long life period and can be found till the end of the September . The study area consists of Shorea robusta forest. Out of the collected specimens Russula species were the dominant all over the study area.

The present study was based on the information available from the questionnaires asked from the local people. Various types of mushrooms were collected. During the collection field note were taken. After the collection, local people were interviewed to get their knowledge about the mushroom. But unfortunately only few people have the idea of the traditional knowledge about the edibility and medicinal value of the wild mushrooms.

Out of 38 species, 11 species were studied in details which were Amanita chepangiana, Auricularia auricula- judae, Cantharellus cibarius, Coprinus atramentarius, Coprinus comatus, Coprinus plicatilis, Dictyophora indusiata,

Macrolapiota procera, Marasmius perforans, Pycnoporus cinnabarinus, Russula emetica. Among them Pycnoporus cinnabarinus, Auricularia auricula-jude were of high medicinal values.

During present study the mushrooms were found to be growing on the different ecological habitat such viz., soil, wood, animal dung, rock and tree trunk. During present study the number of species found growing on soil were 24, wood 7, animal dung 4, tree trunk 2 and rock 1.

Fig. 6: Distribution of mushroom on the basis of their habitat.

4.2 Detailed studies on some collected species.

5.2.1 Amanita chepangiana Tulloss & Bhandary

Fruit body medium to very large, cap 10-26 cm wide, hemispherical to convex, plane with age, white to cream white, sometimes pale lemon yellow in the middle, dry, but glutinous when wet, glabrous, margin tuberculate-striate, up to 3 cm thick towards the middle. Gills free, of various lengths, up to 2.3 cm broad, truncate. Stipe

15-24 x 1.5-3 cm, white, with whitish appressed, zigzag arranged fibrils, these soon disappearing, annulus superior, consistency thin. Nature/Use: Edible Habitat: Terrestrial, mostly found in forest dominated by Shorea robusta.

Date of Collection: 2073-3-14

Col. No. : 8

Altitude: 122

5.2.2 Auricularia auricula- judae (Bull. : fr.) wettst.

Common name- Tree ear; wood ear.

Carpophore- 6-10 cm, ear shaped, sessile or with a short attaching peduncle, outer surface sterile, pubescent, with slight venations; inner surface fertile, reddish brown, at first almost smooth then venose, pruinous because of the spores. When drying out it tends to turn violet and become increasingly circumvolute. Flesh soft, gelatinous, slightly elastic translucent, fragile when dry, reviviscent, no particular odor or flavor. Spores white, cylindrical, smooth, 12-17 × 4-7 µ.

Nature/Use: Edible and medicial The specimen collected from the decay log of the Shorea robusta . Habitat –On broad leaf wood Date of collection -2073-04-16 Altitude - 127 m Col.no-22 Distribution –Worldwide (Europe, Japan, China, N. America, Australia, India & Nepal). Place of collection- Bhartapur 5.2.3 Cantharellus cibarius (Fr. : Fr.) Fr.

Cap 2-10 cm, sometimes larger, convex then open and usually funnel-shaped, margin undulate, sinuate, cuticle, extremely thin, transparent yolk-yellow, glabrous. Gills-like hymenial folds, very decurrent, short, anastomosed, ramified, yolk yellow. Stipe 3-6 × 1-2 cm, tapering from top to bottom, full, solid, cap-colored. Flesh compact, quite

fibrous in stipe, pale yellow and odor strong often like apricots flavor sweet. Spores pale yellows, elliptical, smooth, 7-11 × 4-6 µ.

Nature/Use –In edible Specimen collected from the gravel soilly part of the forest Habitat- Beneath coniferous and broadleaf species. Date of collection- 2073-05-08 Altitude - 114 m Col.no- 37 Distribution –Worldwide. Place of collection- Near chure hill

5.2.4 Coprinus atramentarius (Bull.) Re Cap 5-8 cm, soot-brown, lead-gray, persistently marked, brown or brown-ochre, silky and shiny, ovate, obtuse then campanulate with lengthwise grows squamulose at disc, darker, margin recurved when mature. Gills white then blackish brown, deliquescent, free, ventricose, up to 1.5 cm long, edge floccose. Stipe 7-20 ×0.8-1.8 cm white, initially ventricose, fusiform, narrowing in lower part apex, with small brown scales at base, hollow, fibrous in stipe, no special odor or flavor. Spore black, elliptical, smooth 7-11 × 5-6.5 µ.

Nature/Use –Inedible Specimen collected from the cow dung Habitat –In grassy areas Date of collection- 2070-02-27 Altitude - 138 m Col. no- 34 Distribution – Worldwide. Place of collection- Near Argkhanchi Border

5.2.5 Coprinus comatus (Mull. : Fr.) Pers.

Common name – shaggy cap

Cap 4-6 cm, white turning pink at margin then black, cylindrical when young, up to 20 cm tall then camplanulate, cuticle initially. Continuous then quickly breaking up,

soft, broad, imbricate scales, often raised, white ochreous at margin, with top of cap monsquamose, entire into brown- ochre, striate lengthwise, margin often recurved and split when mature. Gills white then pink, finally and quiescent, free, straighy, crowded up to 1 cm long. Stipe 12-25 × 1-2 cm, white then dirty white or tinged pale, narrowing toward top, with enlarged rooting base, white, movable, thin fugacious ring. Flesh white fibrous, stipe soft in cap where it splits, radially, no odor of flavor. Spores blackish, elliptical, smooth 11-13 × 6.7 µ.

Nature/Use: Edible Specimen collected from the animal dung Habitat- Isolated or gregarious in woods, grassland Date of collection- 2073-03-14 Altitude - 110 m Col. no- 7 Distribution – Worldwide. Place of collection- open field on way to paderiya.

5.2.6 Coprinus plicatilis (Curt. : Fr.) Fr.

Cap 1-3 cm, gray-brown, then ash-gray darker at center, first oval-cylindrical the camplanulate expanded, sulcate-plicate radially, almost diaphanous globrous. Gills cream-colored then gray, eventually blackish, gray separated from stipe by a collarium. Stipe 2.5- 7.5 × 0.1-0.2 cm, pale sometimes transparent, cylindrical, smooth. Flesh whitish, extremely thin, no odor or flavor. Spores black broadly elliptical, smooth, 10-12 × 8-9 µ.

Nature/Use-Inedible Specimen collected from the cow dung. Habitat – Solitary in grass, meadows, gardens. Date of collection- 2073-04-02 Altitude -132 m Col. no- 17 Distribution – Europe, N. America & Nepal. Place of collection- Bhaluhi

5.2.7 Dictyophora indusiata ( Vent. Ex Pers)

Egg upto 4 cm in diameter, globose, ovoidal, white or grayish. Carpophore 15-20 × 2.5-3.5 cm, fusiform or cylindrical, barbed toward top, white porous, hollow, head ogival for a short time, then bell-shaped, yellowish under the gleba, white if stripped, with rugose surface, reticulate with apex perforated and delimited by a raised and distinct collar. Veil white, hanging almost to the ground, with wide polygonal chains formed by elliptical strands. Gleba olive-green, mucilaginous, not very fetid. Spores colorless, elliptical, smooth, 3.5-4.5 × 1.5-2 µ.

Nature/Use-inedible Specimen collected from the decay leaf matter in the soil. Habitat –Found in tropical forests Date of collection- 2073-05-16 Altitude – 128 m Col.no-38 Distribution – North America, rare in Europe, Nepal. Place of collection- Near Paderia Bridge

5.2.8 Macrolapiota procera (Scop.) Singer

The height and cap diameter of a mature specimen may both reach 40 cm, a size truly impressive for the fruiting body of an agaric. The stipe is relatively thin and reaches full height before the cap has expanded. The stipe is very fibrous in texture which renders it inedible. The immature cap is compact and egg-shaped, with the cap margin around the stipe, sealing a chamber inside the cap. As it matures, the margin breaks off, leaving a fleshy, movable ring around the stipe. At full maturity, the cap is more or less flat, with a chocolate-brown umbo in the centre that is leathery to touch. Dark and cap-coloured flakes remain on the upper surface of the cap and can be removed easily. The gills are crowded, free, and white with a pale pink tinge sometimes present. The spore print is white. It has a pleasant nutty smell. When sliced, the white flesh may turn a pale pink.

Nature/Use –In edible Specimen collected from the humus rich forest soil Habitat- Beneath the litter.

Date of collection- 2073-03-14 Altitude - 114 m Col.no- 1 Distribution –Worldwide. Place of collection- Near chure hill

5.2.9 Marasmius perforans ( Hoffm.) Fr Cap 0.8-1.2 cm, whitish turning reddish, convex becoming flat and slightly umbonate with striations. Gills whitish, adnate, fairly crowded, connected by venations. Stipe 2- 3 × 0.1 cm, brown turning blackish, pinkish at the top, tough and velvety. Flesh virtually nonexistent, strong odor and flavor of putrid water, with a hint of garlic. Spores white, lanceolate, smooth, 5.5-6 ×3 µ.

Nature/Use –inedible Specimens collected from the root of the herb plant in the Shorea robusta forest. Habitat –On the conifers. Date of collection- 2073-5-26 Altitude - 140m Col. no- 32 Distribution – In tropical belt including Nepal. Place of collection- Paderia

5.2.10 Pycnoporus cinnabarinus (jacq. :fr) karst Carpophores 3-6 cm or more, bracket-shaped, sessile, deep orange red, tending to darken, at first slightly pubscent then glabrous, fairly fugose, with faint zonation toward margin. Tubes 1-3 mm long, blood red leathery, first spongy then suberrose, odor and flavor negligible. Spores white cylindrical, smooth, 5-6× 2-2.5 microns.

Nature/Uses – Medicinal. Specimen collected from the dry branches in the Shorea robusta forest. Habitat –On broadleaf branches and trunks Date of collection- 2073-03-14 Altitude- 136 m Col.no-5 Distribution- Worldwide. Place of collection- Bhartapur

5.2.11 Russula emetica (Sch. : F r.) Pers. Cap 4-10 cm, varies greatly in color, normally bright red with no violet or pale purple or greenish coloration but varieties may be white, pinkish, yellow, ochreous, globose then convex, eventually depressed with cuticle viscid shiny, may become slightly rugous as it dries, easily detachable with red subcuticle. Gills which then slightly cream-colored, fairly distant, thin rounded as stipe, almost free, fat toward margin. Stipe varies length upto 9-10 cm high in the variety longipes, fairy club- shaped at base white. Flesh white, typically red beneath cuticle, fragile, odor slightly fruity to puffball-like, acrid. Spores white, ovoid, warty, very reticulate, 7.5-12.5 × 6.2-9.2 µ.

Nature/Use- Poisonous Specimen collected from the in the Shorea robusta forest. Habitat –Typically in peat bogs in sphagnum always close to conifers. Date of collection- 2073-03-14 Altitude - 121 m Col. no. – 4 Distribution – Australia, Europe, N. America, Japan, India, Sri Lanlka & Nepal. Place of collection- Bhartapur

4.3 Nutrient Analysis

In the present study, two edible mushrooms, one cultivated (Pleurotus ostreatus) and other wild (Scleroderma cepa) were selected for the analysis of nutrient content. Major nutrients such as iron, manganese, calcium, magnesium, sodium, potassium, phosphate and protein content were analysed by using above mentioned methods. The chemical composition of edible mushrooms determines their nutritional value. It differs according to species but also depends on the substratum, atmospheric conditions, age and part of the fructification (Manzi et al., 2001). The study revealed that iron (134.5 mg/kg) and calcium (3352.3mg/kg) content was higher in wild species while Manganese (29.4 mg/kg), Magnesium (2683.8mg/kg), sodium (978.6mg/kg) , Potassium (36284.1mg/kg), Phosphate(18632.8mg/kg) and protein content (26.1%) were higher in cultivated species.

Table 2: Nutrient Analysis of wild edible (Scleroderma cepa) and cultivated edible (Pleurotus osteratus) through Atomic Absorption Spectrophotometer and UV visible Spectrophotometer for phosphate analysis.

Biochemical Parameters Result Wild edible Cultivated edible mg/kg mg/kg Iron (Fe) 134.5 107.6

Manganese (Mn) 22.4 29.4

Calcium (Ca) 3352.3 677.7

Magnesium (Mg) 1894.4 2683.8

Sodium (Na) 418.9 978.6

Potassium (K) 22534.1 36284.1

Phosphate (PO4---) 10211.6 18632.8

Protein (%) 17.23 26.1

4.5

3.5 wild 3 cultivated 2.5

1.5

1 mg/Kg 0.5

Nutrient Parameter Fig. 7: Graph showing nutritional variation in wild and cultivated edible species.

Percentage of protein content

17.23% Wild edible Cultivated edible 26.1%

Fig. 8: Pie chart showing protein concentration (%) in wild edible and Cultivated edible species.

4.4 Indigenous knowledge and therapeutic use

On the basis of the information collected from 40 respondend of Tharu and 40 respondent of Non-Tharu community, 80% of the tharu people were found to be use mushroom as food, 12.5% as medicine and 7.5% of Tharu people don‟t have any idea of food and medicinal value. On the other hand, 57.5% of the non-Tharu community in the study area use mushroom as food, 7.5 % use as medicine and 35% of non-Tharu community do not use mushroom as food and medicine. Thus, the above data reveals that 68.75% of the people use mushroom as food, 10% use it as medicine and 21.25% of people have no idea about the use and importance of mushroom.

Some mushrooms used as medicine are Auricularia auricula for curing wound of the ear, Scleroderma texense for curing fever of woman during pregnancy period, Termitomyces for treatment of blood pressure and Pycnoporous cinnabarinus is used for curing ripe ear.

According to perception of local people, it was reported that the mushrooms with following features are edible or poisonous:

 Mushrooms having annulus nearby the cap are poisonous.  Mushroom species with curved pileus with annulus are also poisonous.  Mushrooms found on fodder plants are generally edible.  Mushrooms that have warts on their pileus and bad odour are poisonous.  Mushrooms that glow at night are poisonous.  Mushrooms that are peeled off easily are edible.  Mushrooms that are eaten by insects and rodents are generally edible.

CHAPTER FIVE

DISCUSSION

The present study has the following perspectives of the study. First was to collect, identify and enumerate the mushrooms found in the Amrite Community forest, second was to conduct detail study of some mushrooms from the study area, third was to get traditional information of the mushrooms from the local people. The local people were interviewed and asked about the way of identification of edible mushrooms. The study area was visited 3 times throughout the growing season of the mushroom with the help of the local people.

Since the study area is the community forest there is less chance of destruction of the vegetation. The study area is the forest dominated by Shorea robusta with Terminalia chebula, Terminalia belarica, Accacia catecheu and has suitable environment for the growth of the wild mushrooms. Out of 38 specimens collected 34 species were identified up to generic level belonging to 16 families. They are Russulaceae, Amanitaceae, Polyporaceae, Boletaceae, Cantherallaceae, Sclerodermataceae, Coprinaceae, Tricholomataceae, Hydnangiaceae Dacrymycetaceae Auriculariaceae, Agaricaceae, Sparassidaceae, Phallaceae, Xyalariaceae and Pannariaceae.

Among 16 families Coprinaceae is the largest family and the genus Coprinus is dominant in the study area. None of the mushrooms collected from the study area were religious. Among the collected specimens only 10 species were studied in details. The species which were collected from the study area were previously reported from the different parts of Nepal by different researchers.

The edible species Auricuaria auricula reported from the present study has been already reported from Manichur (Adhikari, 1976, 1991c); market (Adhikari, 1987); Kalleitar (Bhandary, 1991).

Commonly known as kanya chyau has been reported from the study area but has been already reported from Nagarjun (Pandey, 1976); market & Manichur (Adhikari, 1976) and Gorepani (Bhandary, 1991).

The species Russula delica found on the study area has been already reported from Royal Botanical Garden, Godavary (1515m) (Adhikari, 1996a, Adhikari & Durrieu, 1996).

The species Coprinus comatus reported from the study area has been already reported from Chima goan (Balfour- Browne, 1968), Godavary (Singh & Nisha, 1976c), Khumal (Pandey, 1976), Manichur, Godavary and Phulchoki (Adhikari, 1976, 1987).

Local people usually add vinegar or Timur while cooking mushroom to minimize the poisonous effect of mushroom. Some use garlic and silver spoon or coin to see the toxicity of the mushroom but sometime it does not work. Adding vinegar is a worldwide method to minimize mushroom poisoning. Many mycologist such as Ramsbottom, (1954); Rinaldi and Tynaldo (1972); Purkayastha and Chandra (1985); Bhandary (1984) and Adhikari (2000a) mentioned about the use of vinegar.

According to local people mushrooms that grow on the bamboo and the colorful ones with bad odor are poisonous. Local people go for collecting mushroom like Scleroderma spp., Amanita hemibapha and Termitomyces spp.

The mushrooms survive in nature under favourable condition. When they get suitable environment for the growth of mycelium and dispersal of spores. But in present situation due to several factors (like change in climate, urbanization, deforestation, natural hazards etc.) the species of mushrooms are declining year by year.

During the study period it was found that the mushroom species like Scleroderma texense, Termitomyces spp. are declining. Being saprophytic, obligatory symbionts as well as part of the mycorrhizal association, these macro fungi play an important role in increasing soil fertility in the forest through biodegradation as well as decomposition of the lignocelluloses compounds of leaf litter. So mushrooms are also known as forest cleaner. The litter debris of vascular flora favours the regulation and maintenance of temperature and moisture in the soil for macro fungi.

During the present study area ethnomycological knowledge were noted. The ethnomycological knowledge which were noted during interview with local people were edible and medicinal values of mushrooms. Similar types of ethnomycological knowledge were also noted by Aryal and Budhathoki (2013d). They collected 27 species of the Basidiomycetes belonging to 6 orders, 13 families and 18 genera from

Baunnakoti community forest, situated in the tropical climate. During ethnomycological survey they have not mentioned the way of identification of edible mushrooms.

A total 30 species of mushrooms under 26 genera belonging to 18 families of Basidiomycetes were recorded by Aryal and Budhathoki (2012) from the Karahiya community forest, western Terai, Nepal, which resembles similar to the present study because similar types of species were found in the both study area. It may be due to the similar climatic region i.e. tropical climatic region.

Protein content in Pleurotus sp. has been documented to range between 8.9 and 38.7% on dry weight basis (Bano and Rajarathnam, 1982). Protein content of cultivated edible species (Pleurotus ostrateus) was found 26.1% which is similar to the result obtained by Manikanandan (2011) in same species (30.4%) and 20.5% reported by Jha and Tripathi (2012). In wild edible species (Scleroderma cepa) it was found 17.23% which is comparatively lower than the protein content in cultivated edible species of mushroom. It can be compared with the Protein content in Ramaria botrytis (16.96%) as reported by Giri and Rana (2008). Iron and Calcium content in Pleurotus ostrateus was found 107.6 mg/kg and 677.7 mg/kg respectively, which was lower than that of Scleroderma cepa with the value 134.5 mg/kg and 2252.3 mg/kg for iron and calcium respectively. The above values can be compared with the values obtained by Thatoi and Singhdevsachan (2014) in Pleurotus sp. of Himachal Pradesh i.e (Fe = 124mg/kg and Ca = 240 mg/kg). The reason for high value of Iron in wild species than cultivated species may be the lack of iron in the soil of field on which paddy plants grow. The study area is the foothill of Churia Hills of Arghakhanchi, the high leeching of calcium from the limestone of hills may be accumulated in the soil. This might be the cause of high concentration of Ca in wild edible species than cultivated edible species of mushroom.

Besides, Fe and Ca other values of minerals are higher in the cultivated species. Phosphorous and Magnesium content in Pleurotus ostrateus was found 18,632.8 mg/kg and 2683.8 mg/kg which is approximate (18,500 mg/kg for P and 2172 mg/kg for Mg ) to the result obtained by Bisaria et al. (1987) in cultivated pleurotus sp. on agro-wastes.

CHAPTER SIX

CONCLUSION AND RECOMMENDATION 6.1 Conclusion The present study focused on the specimens of the mushrooms collected from the Amrite community forest, Banganga municipality-17, Kapilwastu. Total 38 species were collected from the study area. Out of 38 species, 34 species were identified belonging to 16 families. Coprinus species were dominant in the study site. The study site is the forest of Shorea robusta.The largest family was Coprinaceae consisting 5 species. According to habitat 24 species were found on the soil, 7 on the wood, 4 on the animal dung, 2 on tree trunk and 1 on rock.

Detailed studies of 10 species were made of different families. According to the local people (Tharu) it found that the mushrooms that are attacked by insects and rodents are edible. Specially, Tharu and other tribal community people go for the collection of species like Scleroderma, besides it they also collect Termitomyces species which they used as a vegetable. The mushrooms with colorful pileus with warts and bad odour are poisonous as perception of local people. The species like volverialla volvaceae, Ganoderma lucidum, Auricularia auricular, Scleroderma and Pycnoporus cinnabarinus are use as medicine by some of the local people.

Nutrient content in cultivated edible (Pleurotus ostrateus) was found more than that of wild edible(Scleroderma cepa) species except Fe and Ca.

6.2 Recommendation

Some of the important macro fungi such as Scleroderma, Termitomyces and Amanita need special attention to be conserved against threat to avoid their unmanaged and unscientific exploitations. Besides, their harvesting should be done in scientific manner rather than using traditional methods. Over grazing, unmanaged trampling and forest fire should be prohibited by the authorized committee such as DFO, Chief and other members of community forest as well as regular supervision and monitoring of community forest should be regulated for the better development of suitable macro and micro environment. This might be helpful in growth and flourish of mushrooms prevailing in this area which may facilitates the conservation and sustainable development of the community.

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Annex - 1 Questionnaire –Mushroom Uses

Informant –

Occupation - Age-

Sex:  Male  Female

Education - ward no./ tole -

Mushroom Uses

Edibility –

Medicine –

Preparation (mixed with other things)?

How often taken?

Poisonous ?

Effect ( Death, Vomitting)

Cure?

Season-

Habitat –

Looks alike –

Uses of relation-

Any other uses

Who collects it (women, children, men) ?

When /Why ?

Do you always pick mushroom when you see it (why-why not) ?

Annex - 2

Qualitative interview questions

Informant:

Date:

Sex:

Age:

Location :

1. How do you learn about mushrooms names and which ones are edible? 2. Do they grow from seeds? How do they grow? 3. Why do some mushrooms grow on the ground, and some on rotting wood? 4. Do mushrooms grow near certain times of year? 5. Do mushrooms grow near certain kinds of tree? Which ones? Why? 6. Can someone grow mushrooms? How? 7. Why are some mushrooms poisonous? 8. Have you ever been poisoned by mushroom consumption? 9. What do you do if one is poisoned by mushroom consumption? 10. How do you know which ones are edible? 11. Why do you eat mushrooms? 12. Do mushrooms have vitamins or nutrition? 13. Do you ever eat mushrooms you do not know? 14. Are any mushrooms used as medicine? 15. Do people in other communities know the same mushrooms? 16. Do men and women know different mushrooms? Why? 17. Who collects mushrooms? why? 18. Religious uses?

Annex 3

Key to the Enumerated Family, Genus and Species

Basidiocarp usually colored , stipe thick and short ,gills attached………………………..Russulaceae 1(a) Mushrooms exudating latex…………………………………………………………………………Lactarius 1(b)Mushrooms not exudating latex……………………………………………………………..2 2(a) Hymenium with sphaerocysts……………………………………..Russula 2(b) Hymenial surface lamellate………………………………………..3 3(a) Lamellae poorly developed……………………..Cantharellus 3(b) Lamellae well developed……………………….4 4(a) Volva present …………………………………Amanita 4(b) Volva absent …………………………………Laccaria 2(c) Hymenial surface enclosed in cavity……………………………….………….5 5(a)Exoperidium splitting into rays………………………………Geastrum 5(b) Exoperidium not splitting into rays…………………………….Scleroderma 2(d) Hymenium lining interior of pits or tubes…………………………………………….6 6(a) Tubes deep or if shallow,sterile on ridges,texture not soft and putrescent………………polypores Basidiocarp with somewhat thickened base and a prominent ring when young with attached gills……………………………………………………..Trichomataceae a. Presence of thin stalk…………………………………Marasmius b. Presence of thin stalk with papery basidiocarp………….Mycena c. Presence of small short stipe or lacking stipe……………...Pleurotus

Photo plates

Pycnoporous cinnabarinus (Jacq.:Fr)Karst. Sparassis crispa

Guepinia spathularia Macrolapiota procera

Russula emitica (Sch.:Fr)Pers. Coprinus plicalitis

Lacaria laccata Coprinus disseminates

Amanita chapengiana Scleroderma taxiens

Buwaldo boletus Auricularia auricula-jude

Polyporus durus (Timm.)Kreisel. Microporus xanthopus (Fr.) Kuntze

Coprinus comatus (Mull.:Fr)Pers. Dictophora indusiata

Dried sample of Pleuratus oestratus and Scleroderma cepa.

Field visit with local ethnic mushroom collector.

Researcher performing nutrient analysis in laboratory