Summary of biologicaldoctoral thesis: Research on species, distribution and intoxicant of Amanitaceae R.Heim ex Pouzar in Highland

Research Objectives: Identify some biological, ecological characterisitics of Amanitaceae in Highland. Analyze intoxicant of some species in Amanita genus.

MINISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADEMY

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-

Name Ph.D Tran Thi Thu Hien

RESEARCH ON SPECIES, DISTRIBUTION AND INTOXICANT

OF AMANITACEAE R.HEIM EX POUZAR IN HIGHLAND

MAJOR: Botany

Code: 9 42 01 11

SUMMARY OF BIOLOGICALDOCTORAL THESIS

HA NOI - 2018

This thesis was fulfilled at Graduate University of Science and

Technology, VietNam

Scientific instructor 1: Assoc Prof Dr Tran Huy Thai

Scientific instructor 2: Assoc Prof Dr Le Ba Dung

Reviewer 1: Prof Dr Pham Quang Thu

Reviewer 2: Assoc Prof Dr Tran The Bach

Reviewer 3: Ph.D Ha Minh Tam

The thesis will be defended in the Graduate University of Science and Technology (GUST) council at Vietnam Academy of Science and Technology (VAST) at …… on… 2018

This thesis may be found at:

- The library of GUST

- National Library of Vietnam

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INTRODUCTION

1 Significance of the research

Fungi were a saprophyte in the ecological enviroment They could extract enzyme to the enviroment to resolute complex molecules into simple substances Thus they play the important role into improve the natural cycle of material circulation, mineralization of organic compounds, freshing the ecological environment, increasing the fertility of the soil, so increase crops and forest trees productivity

The ecosystem in the Central Highlands is diverse with six main ecosystem types, including tropical evergreen closed forest, deciduous subtropical wet forest, deciduous semi-evergreen tropical forest, coniferous mixed bamboo forest, grass and shrub, residential areas Flora and founain this area is very abundant, has many rare and precious species in Red Data Book of Vietnam

Nature condition in Central Highlands is convenient to

development of Fungi and Amanita genus The research on large fungi is

very little, concentrated on midland area In the Central highlands, there are some research in South of Central highlands, the other areas almost has not researched Amanitaceae play the important role in the Fungi as a diversities and special in poison, theses Fungi are highly toxic and easily confused with some edible fungus

The habit of using mushrooms in the wild and from the forest as food is quite common to the people in the locality This areas was difficulties economic areas, living standard of the people is very low, most

of them are poor and dependent forest Thus forest is a sources to provide a food for living, among them mushroom is a the food that people say is a specialty Wild mushrooms was delicious and aromatic with very high nutrients food But it is also unfortunate confusion between the poisonous mushrooms and edible mushrooms

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In the nature, there are alot of poisonous mushroom belong too

other genus susch: Amanita, Galerina, Lepiota, inobybe, Agaricus… eg: The genus Amanita have Amanita verna, Amanita virosa, Amanita phalloides… should be a confusion for people when using wild mushrooms

as food, in fact, there have been many cases of lethal fungal poisoning due

to lack of knowledge about poisonous mushrooms

Inoder to provide the knowledge to the people who understand and

distinguish between the poisonous mushrooms and edible mushrooms is very necessary Thus, I have chosen the topic

“Research on species, distribution and intoxicant of Amanitaceae R.Heim ex Pouzar in Highland”

2 Research Objectives

- Identify some biological, ecological characterisitics of Amanitaceae in Highland

- Analyze intoxicant of some species in Amanita genus

3 Scientific significance of the research

- Research on biological, ecological characterisitics Amanita genus

- Supplement large mushroom list particulary in Highland and Vietnam in general, simultaneously provide data basic for the other research fields

4 Practical significance of the research

Identify the poisonous mushrooms in the nature to prevent fungal poisoning

5 New result of the research

It is the first time to have a research on Amanitaceae, establish the list of species of Amanitaceae in Highland Identify the scienctific name of

23 species among 33 species and uses molecular biology to identify 16 species of this family in the Central Highlands Introduce 15 species as a new record for large fungi list of Vietnam and 8 species could be a new species

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Research on biological, ecological characterisitics Amanita genus

- Establish the multivariate regression equation to focast the

distribution of Amanita species was Tansoxuathien = C + a*l + b*m +

c*h - d*t

- Determined the intoxicant of Amanita sp.1 to causing death of

experiment animal by oral with LD50 was 4750 mg/kg weight

6 Chapter layout of thesis

This thesis is including 159 pages, 12 tables, 37 figures, 1 map and index

The sections of the thesis were: Index, list of tables, list of figures, list of abbreviations, list of charts

Introduction (5 pages); Chapter 1: Overview of publications (30 pages); Chapter 2: Subject, location, content and research methods (23 pages); Chapter 3: Research results (90 pages); Conclustion and recommend (2 pages); List of published by the author related to the thesis (2 pages); Refefences (8 pages); Index

CHAPTER 1 OVERVIEW OF PUBLICATIONS

1.1 Fungi classification system

1.1.1 History of Fungi taxonomy

1.1.2 Basidiomycete and taxonomy system

1.1.3 Trinh Tam Kiet system

Div Basidiomycota R T Moore (1980)

Fam Amanitaceae R.Heinn ex Puozar (1983): 3 genus (+23

syns)

Characteristic: Fruit body is fleshy, easy to putrid Cap is umbrella shape, stalk central attached, easy to separate Spore radiation produce in the gill Gill free Spores glabrescent, no colour

in the microscope, pink when concentrated Young fruit body has cover

by two membrance, scar when adult

* Gen Amanita Pers (1987): Large distriobution, many species

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wererooting mushrooms, some saprophyte

Characteristic of Amanita genus:

- Many colours like: red, orange, yellow

- Cap fleshy, umbrella shape

- Gill large, white or yellow

- Stalk fleshy, central attached, easy separate

- Spore no colour, globose to elippsoidal, glabrescen

- Saprophyte on land

- When the fruit body is immature, young body volva and skirt connect from cap margin to stalk Then tear to ring and volva – these are

particular characteristic of Amanita genus

* Gen Limacella Murrill (1911): including 3 species

* Gen Catatrama Franco-Mol 1991: inculuding 02 species

According to Trinh Tam Kiet, Le Ba Dung, Ngo Anh, Le Van Lieu there are 37 species (among them 33 species was identified and

04 species was not identified) in Amanitaceae family, 12 species has been described

1.1.4 Fungal poisoning from Amanitaceae situation

Mycetism was a toxic effects from eating the intoxicant in the mushroom The symptoms are from disorders digestive to death The toxins contained are secondary metabolites produced by the distinct biochemical in the fungi cell Mushroom poisoning was a result by eating wild mushroom which not exactly identify Mushroom poisoning sometimes happened with experience mushroom picker

Vietnam was a country which had abundant biodiversity in the wold Recently, there are 3000 fungi species had been recorded in Vietnam in which

1800 species lare fungi, among them there are 50 species has intoxicant Most of the poision mushroom was not lethal poision mushroom, but

the lethal poision mushroom almost was Amanitaceae such as Amanita phalloides, Amanita virosa, Amanita verna Amanita species are confusing to

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other mushroom species, specially when young, boddy ovoid has volve is

similar to Volvariella esculenta, Volvariella bombycina or other species Bovista, Lycoperdon…

CHAPTER 2 SUBJECT, LOCATION, CONTENT AND RESEARCH METHODS 2.1 Subject, location, content and research methods

+ In the field: Chisel, knives,plastic bags, cameras …

+ On tha laboratory: the pick, razor, lamen, …

+ Needle, eppendof, aseptic water, analysis scale and other chemistries, some of molecular chemistries of Sigma, Merck, CTAB, Tris base, acid Boric , NaCl, dNTPs, EDTA, 6X orange loading dye solution, Taq Polymeraza, Ethanol, 2-propanol, Acetic acid glacial, Phenol, Chloroform, isoamyalcohol, Agarose and ITS primer

Animal: BALB/c thoroughbred mouse was healthy in animal shelter

of Institute of Biotechnology, Vietnam Academy of Science and Technology Mouse has feed by standard food and free water

ITS primer list (White et al 1989)

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2.1.3 Location

Highland area (Chu Yang Sin National park, Yok Don National park, Ea Sô Nature conserve, Bidoup Núi Bà National Park, Chư Mom Ray National park, Kon Ka Kinh National park)

2.2 Contents

- Investigate and collect the specimen of Amanataceae in the Highland

- Analyse biological, ecological characteristic of Amanitaceae

collected species in Highland

- Identify the species of specimen was collectd and establish the List of Amanataceae species in Highland

- Analyse intoxic of one species of Amanataceae family

2.3 Research Methodology

2.3.1 Collecting method for fungi in the Field work

- Collect Amanataceae specimen by line survey that go through the different representations (broadleaved forest, coniferous forest, mixed forest ) in Highland

- The time to collect specimen: in rainy season (from June to November)

The specimen was collected base on main characteristic of Amanitaceae family

2.3.2 Processing and preservation specimen method

Specimen was preserve in air and cool place Change the cover when wet or dirty Describe the characteristic of species on the field book such: size, colour, cap surface, merge characteristic, spore, stalk, gill, ….the species was not identified or have enough standard sould be soaked

2.3.4.1 Comparative morphologic method

Base on document of Trinh Tam Kiet (2012,2013), Le Ba Dung (2003), Teng (1964), Singer R.(1986), Jiri Baier (1991), Denis R Benjamin (1995)

2.3.4.2 Moleculare identifying method

Some of the species had similar characteristic, thus we use moleculare indentifying method to analyze the distent between them

2.3.5 Toxicity test method

Toxicity test method is to Confirm the toxicity at different doses of the test substance when tested on animals Animalswas divided to other goups, in which group animals has one dose, and increase the dose from one group to another Note the death animals in the group, the dose and number of death animals in the group is an important parameter in Toxicity test method DoTrung Dam, Dodehe Yeo et al (2012), N’dia Kouadio Frédéri et al (2013), Aristide Traore et al (2014)

2.3.6 Determined ecological factors method

Determined ecological factors method (temperature, humidity, light, altitude) by using Tiger Direct HMAMT-110 (USA), TigerDirect

LMLX1010B (USA), GPS Garmine Trex Vista HCx (USA)

2.3.7 Analyze the interrelation of ecological factors method

MS TAT 2009 and Excel software used for statistical processing Statgraphic Centurion XV software to to establish multivariate regression and to analyze the relationship of density, frequency of occurrence of species Mushrooms with ecological factors

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CHAPTER 3 RESULT AND DISCCUSION

3.1 Amanitaceae R.Heinn ex Puzar (1983) morphology

Fam Amanitaceae R.Heinn ex Puozar (1983): 2 genera Fruit body fleshy, easy to putrid Cap is umbrella shape, stalk

central attached, easy to separate Spore radiation produce in the gill

Gill free ,Spores glabrescent, no colour in the microscope, pink when

concentrated Young fruit body has cover by two membrances, scar when adult

Gen Amanita Dill ex Boehm 1760

Including the most poision mushroom distribution in hold the world

Characteristic:

- Many colour like: red, orange, yellow

- Cap fleshy, umbrella shape

- Gill large, white or yellow

- Stalk fleshy, central attached, easy separate

- Spore no colour, globose to elippsoidal, glabrescen

- Spore from 5-7 x 10-12 µm

- Saprophyte on land

- Hole 20 - 300

- Young body Volva and skirt connect from cap margin to stalk

Then tear to ring and volva – these are particular characteristic of Amanita

genus

Gen Limacella Murrill 1911: 03 species rarely distribute in Asia Gen Catatrama Franco-Mol 1991: there are 02 species in the

world

3.2 Amanitaceae species list in Highland

This thesis was investigated, described, identified and established

25 species of Amanita, Amanitaceae (table 3.1)

T

R T

X

R B T

X

RHG LK&L

3 Amanita crocea Quél

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Map of collection Amanitaceae speciemen in Central Highlands

3.3 Key to species of genus Amanita

1 Stalk complete, volva receptacle 2

1 Stalk complete, volva not receptacle 3

2 Stalk collar shape 11

2 Stalk not nectar shape 12

3 Volva bulb shape, stalk ring 4

3 Volva bulb shape, stalk not ring 5

4 Cap and stalk dark yellow to ferruginous, ring collar shape connect to 1/3

stalk above Spore 5-7 x 8-10 µm Amanita flavoconia

4 Cap and stalk dirty white, minute scales in surface, ring connect 1/3 stalk

basal Spore 6-8 x 8-10 µ Amanita concentrica

(II)

Yok Don NP:

12°45′ - 13°10′

N; 107°29′30″

-107°48′30″ E

(VI)

Bidoup Núi Bà

NP: 12°00'00" -

12°30'00" N;

108°35'00"

-108°75'00" E

(III) Chu Yang Sin NP:

120°14′16″ - 130°30′58″ N; 108°17′47″ -108°34′48″ E

(I)

Ea sô NC:

12O53’18”

-13O02’12” N;

108O28’48” -

108O43’54” E

(V)

Chu Mon Ray

NP (Kon Tum):

14°18′ - 14°38′

N, 107°29′

-107°47′ E

(IV) KonKa Kinh NP (Gia

Lai): 14°09′ -14°30′ N; 108°16′ - 108°28′ E

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5 Stalk withou scales 6

5 Stalk scales, cap verrucose smaller than 2 cm 7

6 Cap verucose saller 2 cm 8

6 Cap verucosecó larger than 2 cm 9

7 Cap light brown, spore smaller 9 μm, verucose sparse, concentrate in a central, spore oblong- ellipsoid, 5-6×7-9 μm, endorsperm yellow Amanita excelsa 7 Spore larger than 9 μm 10

8 Cap flat, slightly concave Ring stalk distintc; Spore globose, 7-9 x 10-12 µm, endorsperm seed oil of floating Amanita pilosella 8 Cap tan colour, stalk cylindiric,light whight, fiber system crystal, baffled, ring stalk indistintc, spore 5-8 x 9-12 µmAmanita multisquamosa 8 Cap dark brown, rings on the central of stalk, surface mucusly, spore 6-8 x 8-10 µm Amanita pantherina 9 Cap convex, white, around the cap have folds, fiber system light yellow, wall, spore 6-8 x 9-12 µm, endorsperm green seedsAmanita hesleri 9 Cap umbrella shape, white, scaly white; Stalk white, ring distinct Fiber system crystal, whithout wall, spore 8 - 10m in diameter, endorsperm green without seeds Amanita cokeri 10 Verucose denserly on cap surface, spot many colour: from white to black, ring collar shape distinct, spore 6-9 x 8-11 µm, endorsperm seed oil of floating Amanita sp.DL274 10 Verucose denserly on cap surface, white; Stalk bulge in the middle, Spore 7-9 x 8-12 µm, endorsperm seed oil Amanita abrupta 10 Verucose sparse, spore thick, globose, 6-8 x 9-11 µm, endorsperm yellow to green seeds, Cap light grey-brownAmanita sp DL89 11 Cap capanulate, light grey to brown-grey, stalk cylindric, ring white, scar shape Spore 6-8 x 8-10 µm Fiber system without wallAmanita Phalloides 11 Cap smooth, orange yellow 14

11 Cap is not smooths 13

12 Fruit body conical 16

12 Fruit body capanulate 17

12 Fruit body umbrella shape 18

13 Cap umbrella shape, white verucose on cap surface, spore elipsoid starch granules, 4-6 x 8-10 µm Pore with pink colour, ring closed to cap, fiber system without wall (4-6µm in diameter) Amanita Sp.1 13 Stype cream white 15