The Uptake of Zinc by

Selected Mushroom Fungi

Sandra. J. Chapman. V ' >

June 1994.

Thesis submitted in partial fulfilment of the requirement for the degree

of M.Phil, The Chinese University of Hong Kong, Shatin.New

Territories，Hong Kong.

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Acknowledgments

I would like to thank Professor S.T. Chang for stimulating discussions and

encouragement and Dr J.A. Buswell for his constant support and

enthusiasm. I would also like to thank Dr K.H. Yung for acting as member

of my Advisory Committee and Dr G.M. Gadd for acting as my External

Examiner.

My appreciation extends to the technicians of the Biology Department for

excellent technical assistance, to fellow colleagues for providing enjoyable

working conditions and to my family for their support and encouragement

throughout.

i

Abstract

A.ll biological systems require zinc in small quantities for normal cellular

functioning. Zinc deficiency causes abnormal growth which has been

associated with metalloenzyme disruption. High zinc levels interfere with

metabolism leading to a fungistatic or fungicidal effect. Zinc influences

morphological characteristics such as the conversion from a filamentous to

s yeast like morphology. Volvariella volvacea when viewed

macroscopically on high zinc concentrations had an altered morphology

v.ith a mucilagenous appearence.

The properties of Lentinus edodes as an aphrodisiac has been attributed to

tne level of zinc in the fruit bodies. It is well known that mushrooms are

able to accumulate zinc and other metals in their sporocarps. Studies were

carried out to examine zinc levels in sporocarps of V. volvacea, Pleurotus

sajor-caju and L. edodes grown on different concentrations of zinc. L.

edodes was able to accumulate zinc to a higher level in the stalks compared

to the caps. No accumulation was seen in P.sajor-caju or V. volvacea.

Fungi are capable of growing at higher levels of zinc than most other

organisms. Six mushroom species were compared for their ability to grow

on zinc treated substrate. V. volvacea was outstanding in its ability to resist

t'le toxicity of zinc compared to the other fiingi. The treated mycelium

produced an extracellular protein matrix and an amorphous hyphal surface.

Zinc was internally compartmentalized at regular intervals along the

hyphae and this was presumed to be nuclear zinc precipitation. However,

studies using fluorescence microscopy did not support this theory.

Microorganisms are known to produce metal binding proteins when grown

under deficient conditions. Gel electrophoresis was used to study the

protein profiles of F. volvacea grown under supplemented zinc and

chelated zinc conditions. One protein band was produced after growth on

chelated medium that was not present under zinc supplemented conditions.

It is proposed that the protein is involved in zinc binding.

ii

Contents Page

1.Introduction 1

1.1 History of zinc. 1

1.2 The biological role of zinc. 2

1.3 Zinc toxicosis. 6

1.4 Mechanisms of zinc uptake and transport in fungi. 7

1.5 Bioremediation. n

1.6 Uptake of heavy metals by fruit bodies of edible 13

mushrooms.

1.7 Mushroom fungi selected for study . 15

1.8 Purpose of study. 17

2. Materials and Methods 18

2.1 Organisms 18

2.2 Mtdh 18

2.3 Media chelation 20

2.4 Chemicals 20

2.5 Zinc content of fruit bodies grown on substrates

containing different concentrations of zinc 21

2.5.1 Substrate preparation for V. volvacea

inoculum 21

2.5.2 Cultivation of K volvacea fruit bodies. 21

2.5.3 Cultivation of P.sajor-caju fruit bodies. 22

2.5.4 Cultivation ofZ^. e ioc/以 fruit bodies 23

2.5.5 Preparation of biological material for

atomic absorption spectrophotometry. 24

2.6 Effect of different concentrations of zinc on the

growth of six mushroom ilingi. 25

2.6.1 Radial growth study 25

2.6.2 Biomass study 26

2.7 Microscopic studies of V. volvacea. 27

2.7.1 Coomassie Blue preparation, staining of

V. volvacea hyphae. 27

iii

2.7.2 Dithizone staining of V. volvacea hyphae. 27

2.7.3 Fluorescence microscopy. 28

2.7.4 Scanning electron microscopy. 28

2.8 Preparation and analysis of K volvacea proteins

using gel electrophoresis. 29

3. Results 33

3.1 Zinc Uptake by Fruit Bodies. 33

3.1.1 Uptake of zinc by V. volvacea. 33

3.1.2 Uptake of zinc by P. sajor-caju. 33

3.1.3 Uptake of zinc by L. edodes. 34

3.1.4 Symptoms of zinc toxicity in L. edodes. 44

3.2 Growth studies. 49

3.2.1 Radial growth measurements 49

3.2.2 Biomass measurements. 56

3.2.3 Morphological alterations due to zinc observed

with light and electron microscopy. 63

3.3 V. volvacea staining studies. 73

3.3.1 Protein staining using Coomassie Blue. 73

3.3.Z Zinc staining by dithizone and fluorescence

staining by DAPL 75

3.4 V. volvacea protein profile comparisons

after gel electrophoresis. 81

4. Discussion 83

4.1 Zinc uptake by fruit bodies

4.1.1 Uptake of zinc by V, volvacea

and P. sajor-caju fruit bodies. 83

4.1.2 Accumulation of zinc by L. edodes

fruit bodies and mechanism of toxicity. 84

4.2 Effects of zinc on growth. 88

4.3 V. volvacea mechanisms of tolerance. 89

4.4 Differences in protein profiles of V. volvacea,

grown on different concentrations of zinc. 93

5. Referem二，s.

iv

1. Introduction

1.1 History of Z inc

Zinc was described as far back as 1550 B.C. in the Ebers Papyrus. Zinc oxide was

then used as a medicine to cure skin complaints and now it is commonly prescribed

by dermatologists and is formulated as calamine. It is thought that the Chinese

were the first to extract the zinc metal, but it was described in 1597 by an

occidental traveller and sold as "spialter". The term spelter is derived from spialter

and it refers to less pure zinc. The history of zinc from 1869 to 1994 can be

separated into three phases. The first phase was between 1869 to 1940. In 1869,

zinc was. found to be a vital nutrient. Raulin (1869) discovered that it was essential

for the growth of Aspergillus niger. In 1877 it was found to be present in the

human liver, but its function was unknown. In 1900, zinc was reported to be

essential for algal growth, in 1914 for plant growth, and in 1934 for mammalian

growth. Up to 1940 there was little interest in zinc research for two main reasons;

a) there were problems in designing experiments to measure small quantities of

zinc b) the era of "metal fume fever", which referred to industrial zinc pollution.

This era concentrated on the detrimental effects of zinc as a pollutant rather than

its beneficial effects as a nutrient. The second phase was between 1940 and 1959

and was dominated by the discovery of zinc-containing metalloproteins. Keilin and

Mann (1940) reported bovine carbonic anhydrase as being a zinc metalloenzyme

which contained 0.33% zinc by weight. These authors reported zinc to be

necessary for catalytic activity. Now, more than 200 zinc metalloenzymes have

been described. The third phase began when Wacker and Vallee (1959) reported

RNA contained tightly bound enzymes. Since then, zinc has been found to be

essential for numerous other cell processes other than catalysis.

1

1.2 The Biological Role of Z inc

Zinc plays a multifunctional role in both primary and secondary metabolism and is

involved in several metabolic pathways including glycolysis. This is because it is a

common component of metalloenzymes, such as aldolases, dehydrogenases,

polymerases and proteases. The location of these enzymes may be intracellular,

wall-associated, or extracellular. Foster and Wacksman (1939) reported zinc to

cause a marked increase in growth of, and efficiency of glucose utilisation. Nason

et al (1950) found that zinc deficiency in Neurospora crassa caused a reduction

in alcohol dehydrogenase and tryptophan synthesis. When A. niger was grown in

zinc deficient medium, glucose-6-phosphate and 6- phosphogluconate

dehydrogenases showed reduced activity (Bertrand and deWolf, 1957).

Decreased activity may be due to a number of reasons including requirements for

enzyme synthesis, enzyme structure and function. Rhizopus nigricans for example,

requires zinc for the synthesis of pyruvate carboxylase, but not for enzyme activity.

(Foster and Dennison, 1950). A decrease in the activity of zinc-containing enzymes

is probably the reason for the morphological and chemical alterations seen in all

phyla under conditions of zinc deficiency.

Under zinc deficient conditions, growth is nearly always retarded and, in poor

peasant communities of Iran and the Middle East, dwarfism is an abnormality that

has been associated with poor zinc uptake in the diet. Wegener and Romano

(1963) leported that zinc stimulated growth through a primary effect on RNA

synthesis. It is now known that zinc proteins are involved in the transcription and

translation of genetic material. DNA-dependent polymerisation of RNA is an

essential process for transcription for the formation of mRNA before the

translation process. In yeast RNA polymerase, zinc aids in attaching the entering

nucleotide to the growing RNA chain. (Lattke and Weser, 1977). One or more

2

RNA polymerases which contain zinc are needed by most, if not all eukaryotes.

(Cheblowski and Coleman, 1976). Substantial quantities of firmly bound zinc

stabilise the structures of RNA, DNA and ribosomes (Prask and Plocke, 1971).

Zinc is tightly bound to RNA polymerase II (Pebranyi et aL, 1977). It is needed

for the structural integrity of ribosomes and deficiency causes ribosomes to

dissociate and unfold. Zinc deficiency in microorganisms has been shown to cause

a decrease in protein, RNA (ribosomal) and pyridine nucleotides and this ultimately

leads to impaired cell division and reduced growth and repair (Wegener and

Romano, 1963). In poultry, skeletal abnormalities are a characteristic symptom of

zinc deficiency. Tibial collagenase is a zinc metalloenzyme, the absence of which

causes a reduction in bone collagen synthesis (Starcher et al, 1980). In swine,

parakeratosis is a skin disorder causing hyperkeratinization and lesions that is also

associated with zinc deficiency. Moreover, zinc is associated with maintaining

constant levels of vitamin A. The synthesis of retinal binding protein, which is a

carrier of vitamin A, decreases with a reduction in zinc levels (Smith et al, 1973;

Chhabra and Arora, 1985). Night blindness was associated with decreased retina

dehydrogenase in the liver of zinc deficient lambs (Arora et al, 1973). In

dimorphx fungi, zinc affects morphological differentiation. Yamaguchi (1975)

showed that zinc stimulated the transformation of Candida albicans from a

filamentous to a yeast like morphology. Cell membranes lose their ability to retain

solutes (Carlton, 1954). In Eucalyptus marginata, leaf colour changes from green

to bronze and red areas developed before necrosis and some deficiency symptoms

were associated with increased phosphorous absorption and transport under zinc

deficient conditions (Loneragan et al, 1982). French beans are particularly

susceptible to zinc deficiency. Leaves show light green chlorotic regions, become

deformed and new leaves become dwarfed and crumpled (Vitosch et al, 1973).

3

Photosynthesis is disrupted by zinc deficiency due to the effect on zinc dependent

enzymes e.g. carbonic anhydrase involved in the process (Everson,1970)

Reprodjctive function and developmental abnormalities are common

characteristics of zinc deficiency in all phyla. In fungi, deficiency will prevent spore

germination and in plants there is decreased seed and fruit production.

Influorescence formation, fruit and seed formation can be abortive at several stages

from premature flower dehiscence to ovule abortion (Riceman and Jones, 1958).

In animals, deficiency may cause foetal wastage, birth anomalies, hypogonadism

and testicular atrophy. Testicular atrophy may be associated with reduced

testicular alcohol dehydrogenase which is a zinc metalloenzyme. Zinc is involved in

the production, storage and secretion of hormones. Deficiency affects production

and secretion of hormones related to testosterone, insulin and adrenal

corticosteroids. Testicular hypoflinction affects spermatogenesis and testosterone

production (Somers and Underwood, 1969). Deficiency also hampers metabolism,

and lowers protein and nucleic acid levels in the prostrate and testes. Foetal

abnormalities in alcoholic women have been associated with lower zinc levels.

Materna. zinc deficiency in pregnant rats during embryogenesis caused impaired

learning ability and impaired emotional responsiveness in the offspring.

Zinc has a role in protecting membranes. It stabilises membranes and prevents

morphological distortion of protoplasts and spheroplasts after they have been

transferred to distilled water (Korngold and Kushner, 1968). Zinc is present in the

cell wall and its role is reported to be that of stabilisation (Chvapil, 1973). Gray

and Wilkinson (1965) suggested that zinc interacts between components of the cell

wall and may function to anchor lipopolysaccharides.

4

Zinc is a key metal for the formation of secondary products of metabolism such as

antibiotics and fungal toxins. The production of ergot alkaloids is also regulated by

zinc (Weinberg，1977). Aflatoxins are hepatotoxic metabolites commonly found on

agricultural crops such as wheat, rice and cassava. They can be produced by

Aspergillus flaws or A. parasitica. Zinc is specifically required for the production

of aflatoxin (Marsh et al., 1975; Mateles and Adye，1965). Soyabeans have been

found to have litttle aflatoxin even though they may be colonised by the fungus.

Gupta and Venkitasubramanian (1975) attributed this to zinc being bound to

phytic acid and therefore was unavailable to the fungus. Autoclaving was thought

to increase zinc availability since autoclaved soyabean had higher levels of

aflatoxin. However, Stossel (1986) suggested the seed coat became cracked and

porous after cooking and this resulted in fungal colonisation and aflatoxin

accumulation. Fusarin C is a toxin produced by Fusarium sp which has a

mutagenic effect similar to that of aflatoxin. It is a polyketide which has been

linked v/ith oesophageal cancer. Jackson et al. (1989) found that zinc could

regulate the production of this compound. These authors also reported that zinc

increased ammonium uptake (probably due to the stimulation of the synthesis of

nitrogen containing compounds), carbohydrate synthesis and dry weight in

Fusarium gramimariwi. The mechanism of regulation is unknown. Zinc is a

primary regulator of versicolorin, a polyketide secondary metabolite produced by

Aspergillus parasiticus (Failla and Niehaus, 1986). 3-Acetyldeoxynivalenol (3-

ADN) is thought to be a precursor of deoxynivalenol (vomitoxin) which is an

important mycotoxin produced by Fusarium graminearvm. Vasavada and Hsieh

(1988) found that Mg, Fe and Zn suppressed growth but stimulated 3-ADN

production. Pilz et al (1991) reported that Sclerothm rolfsii required zinc for

growth and biosynthesis of an extracellular, branched B-l,3-B-l,6-glucan. An

increase in zinc led to increased yield of glucan that was inversely proportional to

5

the yield of biomass. Pigment production is also influenced by zinc. White and

Johnson (1970) showed zinc to be a specific factor whose concentration has

important regulatory effects on the growth of Helminthosporium and its pigment

cynodontin while Kalyanasundaram and Saraswathi-Devi (1955) reported that

antibiotic production by Fusarium vasinfectim was affected by low and high levels

of zinc.

1.3 Z inc Toxicosis

Many rr etals are able to disrupt normal cellular functioning. They can block

functional groups of enzymes, cause denaturation of proteins and disrupt transport

systems for essential ions. They disrupt membrane integrity which leads to

increased permeability and subsequent leakage of cellular materials. Zinc toxicosis

is often observed in biological systems and most plant species are damaged in the

range of 200-300 ug/g dry weight of leaves (Balsberg Pahlsson, 1989). Wyn Jones

and SutclifFe (1972) observed an effect of zinc on the influx and efflux of

potassium. Zinc tolerant higher plants take up more zinc than do non-tolerant

plants and the zinc is bound mainly to cell wall sites (Burton and Peterson, 1979).

Lichens are able to accumulate zinc to high levels. Experiments involving Lasallia

papulosa showed the lichen to accumulate zinc up to 2560 ppm when growing in

natural habitats. Interestingly, in the laboratory this lichen exhibited depressed

photosynthesis at zinc concentrations of 428ppm (Nash, 1975). The author pointed

out that the discrepancy may be due to the first set of data representing total

accumulc tion over 70 years and the second data represented 9 days accumulation.

Lambinon et al (1964) described morphological changes associated with high zinc

uptake. The podetia of Cladonia cornutoradiata and C. pyxidata remained small

and were also swollen, twisted, darkened and abnormally granular-squamous. Nash

(1975) showed that after watering Cladonia imcialis with a zinc solution,

6

photosynthetic rates and respiration rates were reduced. High metal accumulation

may have little relevance to phytotoxicity assessment as particulate entrapment

may occur. These particles may not be readily solubilized and thus be of little

metabolic significance even though they contribute to the total metal content.

1.4 Mechanisms of Uptake and Transport in Fungi

There are two recognised processes which result in the uptake of metals.The first

is a rapid, reversible, non-specific, energy and temperature independent process

representing adsorption. The metals are thought to bind to fixed negatively

charged groups on the hyphal surface membrane (Rothstein and Hayes, 1956).

These groups do not discriminate between different divalent cations. Fuhrman and

Rothstein (1968) and Paton and Budd (1972) showed that cations compete for the

non-specific binding sites. Zinc uptake can therefore be reduced by the presence of

other divalent cations. In yeast, binding is thought to occur mainly to anionic

groups of polyphosphate and carboxyl. Metals may also bind to impermeable slime

layers or other matrices. Denny and Wilkins (1987) found high concentrations of

zinc in a mycorrhizal fungus which was growing on a polluted substrate. Most of

the zinc was located extrahyphally and bound to the cell wall or extracellular slime

polymers. Components of the extracellular slime possess electronegative sites that

could bind zinc ions.

The second process occurs after adsorption and is slower, irreversible, highly

specific, is affected by anaerobiosis and is temperature dependent. Uptake is

inhibited by inhibitors of glycolysis and of aerobic respiration, and also by

uncouplers. It requires sustained energy and an intact membrane. Transport into

the cytoplasm across the plasma membrane is selective (Paton and Budd, 1972;

Mowll and Gadd, 1983).

7

There are three hypotheses for energy linkage and cation uptake. 1. Active

transport involving plasma membrane ATPases. 2. Transport driven by ATP or

other phosphates. 3. Zinc uniport driven by membrane electropotential.

1. Active transport involving plasma membrane ATPases. The hydrolysis of ATP is

coupled to translocation across the plasma membrane. Active transport may be

driven by ATP or other phosphates. Paton and Budd (1972) proposed that

polyphosphates were involved in uptake by N.vasinfecta.

2. Transport driven by a plasma membrane proton pump. Bakker et al (1986)

described a close relationship between respiration and membrane potential.

3. Zinc uniport driven by membrane electropotential. Hyperpolarisation of the

membrane may stimulate zinc transport. Stroobant and Scarborough (1979)

described the calcium/ hydrogen ion antiporter in the plasma membrane vesicles of

N.crassc Antiporters may translocate ions across the plasma membrane,

depending on gradients of hydrogen ions and cations.

Failla a，id Weinberg (1977) reported that zinc transport in C.utilis exhibited

saturation kinetics and the cells accumulated zinc against a concentration gradient,

a characteristic feature of active transport. They found that zinc uptake was not

influenced by the amount of phosphate in the growth medium and detected no

rapid change in polyphosphate levels. Paton and Budd (1972) reported

N.vasinfecta to have a single system for the second phase of zinc transport. Zinc

transport in N.vasinfecti was shown to be correlated with membrane potential

(Budd, i989) and two kinetically separable systems were described (Budd, 1988).

8

In system 1, zinc transport was enhanced by zinc deficiency while system 2 was

not. Transportation at all concentrations was greatly enhanced in mycelium with

reduced magnesium content. This is possibly due to zinc entry occurring via an

activated magnesium transporter. Fuhrman and Rothstein (1968) found that uptake

was enhanced in cells that were pretreated with phosphate and glucose while

Paton and Budd (1972) found manganese competitively inhibited phase 2 zinc

uptake. Failla et al. (1976) reported cadmium to be a competitive inhibitor of zinc

uptake in C, utilis. These authors described the translocation of zinc to be via an

energy dependent highly specific saturable process requiring an intact membrane.

This specific transport system transfers zinc into a virtually non-exchangeable pool.

There was no efflux system for zinc and the only way to release it was to treat the

cell in such a way that the cell membrane integrity was altered.

Siderochromes or siderophores are low molecular weight chelators of iron that are

released under conditions of iron deficiency. Bioavailiability of iron in aerobic soils

is restricted by the insolubility of ferric hydroxide. They were first discovered in

1952, wnen ferrichrome and coprogen were discovered. A number of siderophore

families have been identified including ferrichromes, ferrioxamines, flisarinines,

coprogens, aerobactins, catecholamides, pseudobactins and mycobactins (Van der

Helm et al, 1987). Among these groups, the hydroxamates are produced by flingi

and the catecholates by bacteria. Their function is to counter the effect of iron

limitation in the environment or within host tissues. Under conditions of iron

limitatio.i they are involved in solubilisation, transport and storage of the iron.

Siderophores are excreted by most microorganisms into the external environment.

Blue green algae can grow at alkaline pH by producing natural chelators (Lange,

1974). Iron aquisition systems are thought to be involved in the transport of other

essential trace elements especially those metals such as zinc which are required for

9

the activity of metalloenzymes (Visca et al.、1992). Pseudomoans aeruginosa

synthesises two siderophores known as pyochelin and pyoverdin. Pyochelin is

poorly water soluble and is known to form complexes with zinc ions (Cuppels et

a/., 1987). Siderophore production can be affected by the presence of metals in

iron deficient surroundings. Pseudomonas aeninginosa, a plant growth promoting

bacterium, produced pyroverdin provided the medium was supplemented with

zinc, cadmium or nickel. In P. aeruginosa zinc at concentrations of above 500nM

induced the pyoverdin system (Hofte et al.’ 1993). Under iron limiting conditions

the addition of zinc to cultures of Azotobacter vinelandu increased siderophore

production (Huyer and Page, 1988).

Most plants do not produce siderophores but are able to use microbial

siderophores as iron sources (Leyval and Reid, 1991). Many microorganisms are

also able to utilise ferric complexes which they themselves have not produced.

Plessner et al. (1993) found Bradyrhizohium japonicum was able to utilise

ferrichrome (a siderophore commonly produced by fungi) and rhodotorulic acid

which is synthesised by yeasts and fungi. Bacteria therefore are able to compete

with fungi by utilising the siderophores that are produced under nutrient stress.

Eng-Wilniot et al. (1992) isolated five diverse iron sequestering compounds from

the supernatants of iron deficient cultures of vegetative mycelia of the cultivated

mushroom, Agaricus bisporus. Such compounds may play a role in the switch

from vegetative to reproductive growth in mushrooms such as Agaricus bisporous.

It is not known if such a transport system exists for other nutrients such as zinc but

zinc binding proteins have been discovered that may play a role in transportation.

Remade and Vercheval (1991) reported a zinc binding protein in a metal resistant

bacterium A lealjgenes eutrophis. The protein was produced under elevated

10

conditions of zinc and differed from metallothionein. The zinc binding capacity

was assumed to be due to the presence ofcarboxyl groups harboured by the acidic

amino acids and to a lesser extent to the few sulphydryl groups of cysteine

residues. Lawford et al (1980) studied the transport and storage of zinc in C.

磁s grown in zinc deficient medium. The ability to hyperaccumulate zinc is

determined by the growth phase and the content of intracellular zinc. It is not

known how the cellular zinc concentration influences the capacity of the cell to

sequester large amounts of metal from the medium. The unidirectional nature of

zinc catbn transport in yeast may be due to a zinc sequestering molecule. Failla et

“/. (1976) described the existence of an inducible cytoplasmic zinc-binding /storage

protein which had metallotheionein-like properties. Such proteins play an

important role in organisms that require different quantities of metals during

different periods of their life-cycles, or for the production of primary and

secondary metabolites. Yeast cells exhibit marked differences in their zinc content

at different times during the growth cycle (Failla and Weinberg, 1977). In fungi,

biological roles of zinc include those related to cell replication and differentiation

(Hambidge et al, 1986). Elevated zinc levels would be required at these times

1.5 Bioremediat ion

Non-biodegradable metals are constantly being discharged into the environment

from various industrial sources. These metals are toxic to living organisms and are

a serious threat to public health. Metals from industry should be removed at the

source but usually are not and so have to be removed from the environment.

Conventional methods of metal removal include chemical precipitation,

electrolysis, ion exchange, solvent extraction and evaporative extraction but they

have limited application in industrial situations. These techniques cannot be used

on a routine basis because of high cost. Some fungi however are able to tolerate

11

and take up high levels of metals and their use for removal purposes could

provide ‘1 convenient alternative to conventional methods of metal removal. Both

living and dead biomass can be used for metal bioaccumulation (Fourest and Roux,

1992). Leuf et al (1991) showed that Aspergillus sp can remove zinc from

polluted water. The uptake of metals can be influenced by pH and this has been

demonstrated in numerous fungi. Uptake and binding of Zn by Penicillium

digitatum was inhibited by an increasingly acidic pH (Babich and Stotzky, 1977).

Adsorption is a function of pH and was increased by adding sodium hydroxide to

the medium. Schinner and Burgstaller (1989) used a Penicillium sp to leach zinc

from industrial wastes and mixtures of metal oxides. Sanglimsuwan et al (1993)

tested the uptake of zinc, cadmium and copper by Plenrotus ostreatus. There was

an increase in the uptake of the zinc ions which was proportional to an increase in

the media. Pleurotus ostreatus took up about 7% of the copper, zinc and cadmium

ions. Mixed cultures of Aspergillus niger and Trichoderma harzianum were able

to oxidise the sulphides of copper, lead and zinc to sulphates. With the exception

of cadmium, the sulphide particles in the medium were adsorbed to the surface of

the mycelium (Wainwright and Graylson, 1987). Under conditions of metal

toxicity, intracellular uptake may be linked with increased membrane permeability

and resultant exposure of metal binding sites. Various treatments can increase the

capacity of fungal biomass for adsorption. The use of detergent improved the

removal of copper by Pemcillhim spimdosiim, presumably by exposing more

copper binding sites. Agariais bisporus has been shown to accumulate Ag and Hg.

Silver-protein complexes were recorded which may correspond with

metallotliioneins or analogous metal-binding proteins (Byrne and Tusek-Znidaric,

1990).

12

A number of basidiomycetes growing in polluted environments show elevated

concentrations of metals in their fruit bodies. The use of sporophores in assessing

absorption is practical but has limitations. The metal concentration in the

sporophore may not be the same as in the mycelium and so sporophore levels can

only be used to reflect the capacity of the mycelium to absorb metals from the

substrate.

1.6 Uptake of Heavy Metals by Frui t Bodies of Edible Mushrooms.

Some metals such as lead, cadmium and mercury are toxic to living organisms

whereas other metals such as chromium, copper and zinc are essential

micronutrients and are needed for normal growth and cellular activities. Although

they are essential micronutrients, in large quantities they can be toxic to the

mammalian system (Bowen, 1966). The magnitude of toxicity reflects the quantity

of contaminated material consumed. By measuring the average daily consumption

of a foodstuff，the metal content and the daily recommended intake levels of a

particular metal, it is possible to analyse its qualities as a potential health hazard.

The recommended daily intake of zinc is 15mg per day (Orten and Neuhaus,

1984). Edible mushrooms were analysed for lead by Kalac et al. (1991) and it was

concluded that mushrooms should not be consumed within a radius of 1km from

the source of pollution. The main sources of heavy metals are smelting factories

and busy roads. Busy roads may have elevated zinc levels nearby due to the

presence of zinc oxide in car tyres.

Tyler (1982) studied the uptake of metals by mushrooms in relation to the

properties of substrate. The metal concentration in sporophores of the litter-

inhabiting Collybia peronata and the soil inhabiting Amantia rubescens were

analysed and compared to the concentrations of the metal in the substrate. Cd, Rb,

13

K，Cu, and As accumulated in both flingi compared to levels in the substrate

whereas Na, Pb, Fe, Al and Mn and Ca were excluded. Zn and Mg showed no

differences in distribution. It was concluded that specific uptake is more a function

of fungal species than of substrate properties. Vogt and Edmonds (1980)

compared concentrations of nutrients in four diverse ecosystems and fruiting

bodies found there. There were no strong differences in nutrient accumulation

patterns between flingi in the same genera collected from different ecosystems.

However, Lepsova and Mejstrik (1988) reported that metal concentrations in fungi

depended upon the physiology of the species, particularly on its trophic pattern.

Tyler (1980) analysed 130 species of Basidiomycetes and reported that

bioconcentration occurred but only to a limited extent. The extent was depicted by

taxonomic status or by soil/substrate properties.

Vogt and Edmonds (1980) found that different genera and species of fungi showed

wide variations in their capability to concentrate metal nutrients within their

fruiting jodies and reported concentrations ranging between 15-278ppm. Tyler

(1980) found species of Polyporus had low zinc, species of Agaricus and

Lycoperdaceae contained relatively high amounts. Bioconcentration was measured

only in nygrophoriis mtratus (1025|.ig/g) and bioexclusion in Polyporus gibbosus

(9ug/g). Hinneri (1975) surveyed 90 macroflmgal species and found that members

of the Agaricaceae contained up to 200ppm zinc, Otidea onotica 1200ppm, and

Calocen’ viscosa only 35ppm. He pointed out that for higher plants the zinc

content is about 20ppm and therefore the average content for fungi is relatively

high. Tyler (1980) found sporophores to be rather rich in zinc (median lOOgg/g)

This leviil is a little higher than most vascular plants and about the same as mosses

and lichens.

14

McCreight and Schroeder (1977) analysed lead, cadmium and nickel in

Lycoperdon perlatum and found no differences in metal levels in washed and

unwashed sporophores. The peridium, internal gleba and base were analysed . Low

levels of metals were found in the gleba and base, indicating the metals were on or

in the peridium. If they were located on the outer surface, the metals were not

easily washed off. There was no trace of cadmium or lead in the base indicating

that the source of internal metal was via aerial deposition and penetration via the

peridium rather than the soil.

1.7 Mushroom Fungi Selected for Study.

Mushrooms are a good source of protein, containing high levels of essential

amino acids and are rich in leucine and lysine, unsaturated fatty acids, carbohydrate

and vitamins. Their mineral content is higher than that of meat and fish and almost

twice that of vegetables. Their nutriative value almost equals that of milk (Chang

and Miles, 1989). Although mushrooms cannot replace meat they can serve as a

good substitute in preventing protein malnutrition.

The six fungi chosen for this study were selected because they are edible

mushrooms which, in nature, have adapted to growing on different substrates i.e.

wood, straw, and in the soil.

Lentinus edodes (shiitake mushroom) colonises wood. For centuries the fungus

has been associated with longevity and is highly valued for purported aphrodisiac

properties. It is also reported to have anti-tumour activity, to stimulate the

production of interferons and to have beneficial effects in cases of cardiovascular

disease. Both these functions have been linked to the levels of zinc in the fruit

15

bodies. Analyses of the fruit bodies of the shiitake by workers in Japan and the

United States have revealed marked differences in the levels of zinc which are

thought to be due to the use of different growth substrates. Levels varied from

54.7ng/g to 5.5|Lig/g.

Pleurotus sajor-caju is a wood colonising fungus. It is able to grow over a wide

range of temperatures (20-35°C) and on various types of lignocellulosic waste and

crop residues. It has been reported that Pleurotus sporophore zinc levels can be

higher than the substrate, which implies that these mushrooms have a tendency to

accumulate zinc (Bano et al, 1981).

Volvarie/Ia volvacea colonises straw. It is traditionally grown on paddistraw but it

is able to grow on rice straw, dried banana stalks, banana leaves, wheat straw,

water hyacinth and sugarcane bagasse. The use of cotton waste as a substrate

gives a higher yield and a regular cropping of Volvariella volvacea (Yau and

Chang, 1972)

Flammulina velutipes, commonly known as the winter mushroom forms fruit

bodies at low temperatures. It is a wood colonising fungus which is commonly

grown on the sawdust of most conifers.

Ganoderma is a medicinal fungus which has been used by the Chinese for more

than two thousand years. The mushroom is reported to have antitumour activity

(Miyazaki and Nishijima, 1981; Mizuno et al., 1985). It is thought to improve

intelligence by enhancing memory, hearing, vision and smelling and retard ageing

and prolong the life span.

16

Tricholoma matsutake forms a mycorrhizal association with plant roots.

Mycorrhizal fungi can enhance the uptake of trace amounts of micronutrients such

as zinc to the plant host. Bowen et al (1974) showed that zinc absorption was 1.4

times higher for plants with associated ectomycorrhizal than for uninfected plant

roots.

1.8 Purpose of Study

The Objectives of the Present Study were to:

a) Examine the effects of zinc on the growth of the six selected mushroom fungi.

b) Determine zinc levels within different parts of the fruit bodies of mushrooms

following growth on substrates containing different levels of zinc.

c) Investigate the effect of zinc on hyphal morphology.

d) Identify specific mechanism(s) for zinc uptake by V.volvacea.

17

2. Materia ls and Methods

2.1 Organ isms

The following organisms were studied:

a) L. edodes. Strain L54.

b) P.sajor-caju Strain P27

c) V. volvacea. Strain VV34.

d) Ganoderma sp. Strain G-1

e) Tricholoma sp. Strain T-2

f) Flamnnilina sp. Strain Fl-2

All strains were obtained from the culture collection of the Biology Department at

Chinese University of Hong Kong. Fungi were maintained at 4°C (15°C in the case

of V.voh^acea) on Potato Dextrose Agar (PDA) slants and subcultured every three

months.

Unless otherwise stated V. volvacea was routinely cultured at 32°C and the other

fungi at 25。C.

2.2 Med ia

The composition of the defined growth medium was as follows:

g/1

KH2 P O 0.2

MgS04 .7H20 0.05

CaCl2.7H20 0.013

2,2-dimcthylsuccinate (DMS) 1.46

Glucose 10.0

L-asparagine 1.0

NH4 N03 0.5

Mineral solution^ 1.0ml,

Vitamin solution^ 0.5ml.

The final pH was adjusted to 5.5 with 2M KOH.

18

a Mineral solution (added immediately before filter sterilization to prevent

precipitation by phosphate.)

g/1

MgS04 .7H2 0 3 .0

MnS04 .H2 O 0.5

NaCl 1.0

FeS04 .7H2 O 0.1

C0SO4 0.1

CaCl2 0.082

CuS04 .5H2 O 0.01

A1K(S04 )2 0.01

H3 BO3 0.01

NaMo04 0.01

ZnS04： appropriate volumes of stock solutions containing lOOOppm and

10,000ppm zinc as zinc sulphate were added to the basal medium as indicated.

Actual zinc concentrations in the medium were determined by atomic absorption

spectrophotometry.

^Vitamin solution

ing/l

Biotin 4

Folic acid 4

Thiamine HCL 10

Riboflavin 10

Pyridoxine/HCL 20

Cyanocobalamin 0.2

Nicotinic acid 10

DL-Ca Pantothenate 10

P-aminobenzoic acid 10

Thioctic acid 10

pH4.5

19

2.3 Med i a chelation

Standard grade chemicals purchased from commercial sources contain trace

quantities of zinc. In order to exclude contaminant zinc from the medium,

puratronic grade 1 trace elements were used and all other components were

chelated with Chelex 100 resin. All glassware was acid washed with 6N HCl and

rinsed six times with double distilled water.

2.4 Chemicals

Chemicals used in this study were from standard commercial sources except for

minerals which were putratonic grade 1 chemicals purchased from Johnson

Matthey. UK.

20

2.5 Z i nc Content of Fru i t Bodies Grown on Substrates Contain ing Different

Concentrat ions of Z inc .

2.5.1 Substrate .^reparation for V. volvacea Inoculum.

Cotton waste (2.35kg) and 48g lime were mixed together in a large bowl which

contained 3600ml water. During the preparation, the cotton waste was teased

apart to ensure uniform mixing. Samples of 400g were added to autoclavable

plastic bags. Plastic eye-glass holders were fitted around the bag openings, the

edge was fanned out around the holder and the space in the centre filled with a

cotton wool bung. Fifteen bags were prepared in this way and then autoclaved for

at least one hour at 121°C ISpsi. After cooling, the contents of the bags were

inoculated with a five day old culture of V. volvacea grown on potato dextrose

agar (PDA) in 9cm petri dishes. The agar and fungus were cut into squares 2 cm x

2 cm and each bag inoculated with five squares which were spaced out evenly over

the surface of the substrate. The bags were then incubated for fourteen days at

32°C to allow the fungal mycelium to penetrate into the cotton waste medium.

2.5.2. Cultivaticn of V. volvacea Fruit Bodies.

Six bowls, containing various quantities of zinc sulphate were each filled with 7.8

liters of water. When the zinc had flilly dissolved, 104g of lime and 104g wheat

bran were mixed together and added to the water. Cotton waste (4.9kg) was then

added to each bowl, dampened and teased apart in the mixture for 2 hours to

ensure even distribution of the zinc. The contents of one bowl were distributed

between four baskets and a representative sample of cotton waste taken from each

basket for subsequent analysis using atomic absorption spectrophotometery.

21

The substrate was subjected to a pasteurization process to destroy unwanted

microorganisms. Baskets were steamed at 62。C for 3 hours and then at 52°C for 5

hours with ventilation.The temperature was then reduced to 35。C and the humidity

maintained at 90% before spawning was carried out. The bags containing the

inoculum were opened and half the cotton waste in one bag used to seed one

basket. Inoculum material was broken up into small pieces and spread evenly over

the surface of the cotton waste in the basket. Some pieces were pushed into the

bed of cotton waste. Once evenly spread, the substrate was flattened to ensure

close contact with the inoculum. Spawning commenced with the control substrate

(no added zinc) and terminated with the highest zinc treated substrate to ensure

there was no zinc carried over from a high treatment to a low treatment. Each

basket、\as covered with a polythene sheet to maintain a constant environment.

After 4 clays, the sheet was removed and the baskets sprayed lightly twice a day

with a very fine spray of water. Approximately 30ml tap water was sprayed each

time onto a basket. Pinheads formed after 5 days and button stages appeared after

7 days. After 9 days the first mature fruiting bodies had formed and others matured

within the next four days.

2.5.3 Cultivation of P. sajor-caju Fruit Bodies.

Seven bowls, containing various quantities of zinc sulphate were filled with 1.21

water. After the zinc sulphate had fully dissolved, 16g lime and 784g cotton waste

were mixed thoroughly in the water. After mixing for at least 30 minutes, 400g of

the substrate was weighed into plastic autoclavable bags. A lOg sample of cotton

waste W IS taken randomly from various parts of each bag and was analysed using

atomic absorption spectrophotometry. A plastic eye glass holder was fitted around

22

the opening of the bag, the edge of the bag was fanned out around the holder and

the cenlie was filled with cotton wool. All bags were autoclaved for 2 hours at

121。C. A 7-day old culture of P. sajor-caju grown on PDA was cut into small

squares (2cm x 2cm) and the bags inoculated as previously described for V.

volvacea. After the bags were incubated at 24°C for 25 days, a circular incision

was made 5cm from the base and the bag was cut away from the cotton waste. At

this stage primordia could clearly be seen. Mature fruit bodies formed after 2-3

days. All biological material was then prepared for analysis by atomic absorption

spectrophotometry.

2.5.4 Cultivation of L edodes Fruit Bodies

Eight bowls, containing various quantities of zinc sulphate were each filled with

1.95 liters of water. After the zinc sulphate had dissolved, 845g sawdust, 260g tea

leaves emd 195g wheat bran were mixed together and added to the zinc sulphate

solution After thorough mixing, the contents of a bowl were distributed between 5

autoclavable plastic bags. Each bag contained 65Og wet substrate. Representative

samples of substrate were taken from each bag and they were analysed using

atomic absorption spectrophotometry. The bags were autoclaved and inoculated

with a 10-day old culture of L edodes grown on PDA. The agar plus mycelium was

cut into square blocks and the bags were inoculated as for V. volvacea. The bags

were plugged and incubated for 60 days at 25°C. They were then transferred to

4°C for 4 days, opened and incubated at 18°C and 80-85% humidity. After

opening, the 40 bags were sprayed with a fine spray of water four times for 5

minutes. Pinheads formed the following day. The bags were then lightly sprayed

for approximately 30 seconds with a fine spray of water three times a day. Mature

fruit bodies were harvested four days later. Fruit bodies were collected from the

control bags first and then from bags treated with increasing levels of zinc to

23

ensure no cross contamination of zinc. Stalks were separated from the caps and all

traces of substrate were removed from the base of the stalks. Samples were

transported in plastic bags for immediate weighing and subsequent drying before

acid digestion as described previously. The remaining bags were left without

watering and a second harvest was obtained five days later. These fruit bodies

were treated in the same way as those from the first harvest.

2.5.5. Preparation of Biological Material for Atomic Absorption Spectrophotometry

Cotton waste and sawdust samples were weighed, immediately dried at 60°C for

2 days aad then at 105°C until a constant weight was attained. A sample (3g) was

transferred to an acid-washed crucible and a white ash was obtained by flirnacing

for 6 hours at 600°C. The ash sample was acid digested by heating to 60°C, adding

5ml nitric acid and then 2ml hydrogen peroxide. After the sample had completely

dissolved, the contents of the crucible were transferred to a volumetric flask and

the volume was made up to 50ml. Samples were filtered through a glass filter,

Whatman C grade prior to analysis. Fruit bodies from one basket or bag were

harvested together as one replicate. The stalks were separated from the caps and

both were immediately weighed. After drying, samples were flirnaced and acid

digested using the same method as that described previously for cotton waste. All

samples were analysed using an Hitachi atomic absorption spectrophotometer.

24

2.6 Effect of Different Concentrations of Z inc on the Growth of Six Mushroom

Fungi .

2.6.1 Radial Growth Study

A slurry ofChelex 100 resin (25g) in 0.2M acetate buffer (pH5.5) was packed into

a glass column (4cm x 35cm). Medium consisting of 2,2-dimethylsuccinate (3.5g),

glucose (24g), L-asparagine (2.4g) and vitamins (1.2ml) in 280ml double distilled

water was passed through the column to remove metal contaminants. Solutions of

spectroscopically pure minerals were added to the chelated medium to the

concentrations shown and the solution filter sterilised The final volume was 360ml.

Aliquots (30ml) were mixed with solutions of agar (4g agar in 170ml double

distilled water previously sterilized by autoclaving). Several batches of this medium

were prepared and the zinc concentration of each batch adjusted as indicated by

the addition of different amounts of Grade 1 zinc.

Plates were inoculated in the centre with a 4mm disc taken from the growing edge

of cultures of the six test fungi grown on PDA. Inoculum cultures were all 5 days

old except L. edodes and Ganoderma sp. which were 8 days old. The plates were

sealed with parafilm and incubated at 32°C in the case of V.volvacea and 24°C in

the case of other mushroom fungi. Radial growth was measured at six equi-distant

points at periodic intervals and the average used to assess fungal growth rates.

Unless stated otherwise values reported are the mean of three replicate cultures.

Micrographs of the growing hyphae were taken using a Nikon light microscope

fitted with phase contrast and a camera attachment.

25

2.6.2 Biomass Study.

All glassware was acid washed and rinsed six times with double distilled water.

Medium consisting of 2,2-dimethylsuccinate (S.llg), glucose (35g), L-asparagine

(3.5g) and vitamins (1.75ml) were passed through a column containing Chelex

resin (25g) as described previously. Spectroscopically pure KH2PO4 and I

NH4 N03 were pipetted into the chelated medium. The pH value of 5.5 was

confirmed and the medium filter sterilized.

Spectroscopically pure minerals, taken from respective stock solutions were

pipetted into 150ml acid washed flasks and double distilled water added prior to

autoclaving at 121° C, ISmins. After autoclaving, 1.5ml of the chelated medium

was pipetted into each flask. The zinc concentration of the medium was adjusted

as indicated by the addition of Grade 1 zinc. Treatments were set up in triplicate,

ranging from Oppm to lOOOppm. The final volume per culture flask was 25ml.

All mushroom species were grown on PDA for 5 days except for L. edodes and

Ganoderma sp cultures of which were 8 days old. A sterile 4mm cork borer was

used to cut a disc of mycelium from the growing edge of the inoculum culture.

After inoculation, the flasks were incubated without agitation. The number of days

of incubation was determined by the period of linear growth ascertained from the

agar plate experiments.

Mycelial biomass was determined by filtering the flask contents through a

Whatman Nol filter paper, drying the filters at 70°C for 12 hours, weighing and

subtracting the original weight of the filter paper.

26

2.7 Microscopic Studies of V, volvacea

2.7.1 Coomassie Blue Staining o^V. volvacea hyphae.

Coomassie Blue, which stains specifically for protein was used to stain 4-day old

mycelium of V.volvacea grown on supplemented (lOOOppm) and non-

supplemented agar medium. Coomassie Blue G-250 (lOOg) was dissolved in 50 ml

absolute ethanol and 100ml 85% (w/v) phosphoric acid added. This solution was

then diluted to 1 litre. Five ml of staining solution was pipetted carefully onto

V.volvacea mycelium. Micrographs of hyphal staining after 4 days growth were

prepared as described above in section 2.6.1.

2.7.2 Dithizone Staining of V.volvacea Hyphae.

Dithizone forms complexes with cations derived from metals grouped in the centre

of the periodic table and produces a characteristic colour change in the presence of

zinc. In vitro studies were carried out to test the specificity of dithizone for zinc

ion. Saturated solutions of 11 trace elements were prepared and 10|al of each

solution added to individual wells in a microplate assay dish. Filtered saturated

dithizone in 60% alcohol (100|al) was added to each well.

Medium was prepared and inoculated as described in section 2.6.1. An acid

washed cover slip was sterilised in alcohol and inserted into the agar at various

distances from the inoculum depending upon the growth rate of the fungus at the

zinc concentration being tested. After the mycelium had grown up onto the

coversH【’ the coverslip was removed and stained with 50|al of a filtered, saturated

stock solution of dithizone in 60% ethanol. The coverslip was then inverted onto a

glass microscope slide and the flingal mycelium viewed using a Nikon light

microscope fitted with phase contrast and a camera attachment.

27

2.7.3 Fluorescence Microscopy.

Hyphae were stained with 4',6-diamidinio-2-phenylinodole (DAPI) as follows. Ten

microlitres of a 1.0|,ig/ml solution of DAPI was pipetted along the edge of the

same coverslip onto which fungal mycelium had been grown as described above.

The stain was then drawn across the specimen using tissue paper as absorbant.

After 5 minutes, the samples were viewed with a Nikon fluorescence

photomicroscope using standard light optics. Standard light optics were used to

detect zinc staining followed by U.V. excited fluorescence to observe the nuclei.

2.7.4 Scanning Electron Microscopy.

Medium was prepared as described in section 2.6.2 and zinc was added to the

growth Jasks to give final concentrations of Oppm and lOOOppm. Two 150ml

flasks containing 25ml chelated DMS medium, without added zinc were inoculated

with fungal growth from a 4 day- old culture of V. volvacea. After 3 days, the

fungal mycelium was harvested, washed 4 times using double distilled water and

blended in 40ml double distilled water for 3x10 second periods. Aliquots (0.5ml)

representing 1.2mg mycelial dry weight were pipetted into each growth flask.

Fungal n‘ycelium was harvested after 12 days incubation at 32°C, transferred onto

a clean coverslip, dried, and then fixed in 3% glutaraldehyde in buffer for Ihour.

The following reagents were prepared.

Cacodyl-ite buffer (0.2M):

Na2(CH3)2 ASO2.3H2O 42.8g

Distilled water 1000ml

28

Hydrochloric acid (0.2M):

Concentrated HCl 16.6

Distilled water 1000ml.

A buffer solution at pH 6.4 was prepared by mixing 50ml cacodylate buffer with

36.8ml hydrochloric acid and 13.2 ml distilled water.

The fungal mycelium was washed in buffer for 5 minutes before immersing in 1%

osmium tetroxide for 2 hours, then washed in buffer for a ftirther 5 minutes. Partial

dehydration was carried out by immersing the sample in increasing concentrations

of ethanol: 50% for 10 minutes, 70% for 10 minutes, 85% for 10 minutes, 95%

for 15 minutes and twice in 100% ethanol for 15 minutes each. After complete

dehydration, critical point drying was carried out in liquid carbon dioxide and the

samples coated with gold. Mycelium was attached to a copper stud using doltite

paint and viewed under a Joel scanning electron microscope, model JSM 5300.

2.8 Preparat ion and Analysis of V. volvacea Proteins using Gel

Electrophoresis

Glassware was add washed and rinsed six times with double distilled water. 2,2-

dimethylsuccinic acid (0.73g), L-asparagine (0.5g), vitamins (0.25ml) and glucose

(5g) were dissolved in 30ml distilled water. The pH was adjusted to 5.5 and the

solution chelated as described previously. After chelation spectroscopically pure

KH2PCM and NH4 N03 were added, the pH was confirmed and the medium filter

sterilised into a 50ml acid washed autoclaved bottle.

29

Twenty 150ml acid washed flasks containing spectroscopically pure minerals in

double distilled water were autoclaved at 121。c ISmins. After autoclaving, 1.5ml

of the chelated sterile medium was pipetted into each flask. Four replicates of zinc

treatments were prepared at Oppm, lOppm, 20ppm, and 50ppm by pipetting the

required quantities of an autoclaved stock solution of zinc sulphate into the flasks.

The final volume of medium per flask was 25ml.

V.volvacea inoculum was prepared as described previously in section 2.7.4. After

incubating at 32°C for 6 days the replicates from each treatment were mixed and

ground under liquid nitrogen into a powder using a mortar and pestle. Samples

were transferred to Eppendorf tubes and placed on ice. The contents of each tube

were then centrifbged at 1 l,600g for 10 minutes. The supernatant was transferred

into an Eppendorf tube and the protein content was determined using Lowry's

assay method Lowry et al. (1951). Samples were then diluted at least 1 in 4 with

sample buffer and heated at 95°c for 4 minutes. The sample buffer was prepared as

follows:

Distilled water 4ml

0.5M Tris-HCl pH6.8 1.0ml

Glycerol 0.8ml

10% (W^V) SDS 1.6ml

2-mercaptoethanol 0.4ml

0.05% (w/v) bromophenol blue 0.4ml

A mini-PROTEAN II Bio-rad dual slab cell was used to analyse protein samples.

A glass plate sandwich was prepared with the separating gel in the centre.

Polyacrylamide separation gel (10%) was prepared as follows:

30

Distilled water 4.02ml

1.5M TrisHCl pH8.8 2.5ml

10%(w/v) SDS lOOpil

Acrylamide/Bis (30%) 33,3ml

10% Ammonium persulphate 50ml

TEMED 5 ml

The stacking gel was loaded on top of the separating gel. A plastic comb was

inserted and the gel set around it to form wells into which the samples were added.

Stacking gel was prepared as follows.

Distilled water 3.05ml

0.5M Tris-HCl pH6.8 1.25ml

10% w/v SDS 50|al

Acrylamide/Bis (30%) 0.65ml

10% Ammonium persulphate 25|il

TEMED 5 III

The comb was removed after the gel had set. Running buffer was added and IS^ig

protein per sample loaded into the wells.

Stock Electrode (Running) Buffer was prepared as follows:

Tris base 9g

Glycine 43.2g

31

SDS 3g

Increase volume to 600ml with dH20 and dilute 60ml stock with 240ml dPLO for

one electrophoretic run.

The gel was run at 200 volts for approximately 30 minutes until the dye front had

reached the bottom of the separating gel. It was then stained with 0.1% Coomassie

Blue R-250 in 40% methanol and 10% acetic acid. After 30 minutes the gel was

destained in 40% methanol with 10% acetic acid for at least 2 hours.

32

3. Results •；..

3.1 Zinc uptake by Fruit Bodies.

3.1.1. Uptake of Zinc by V. volvacea. I

Figures 1-3 show the mean zinc concentrations in cotton waste substrate and fruit

bodies harvested after 9 days. Values are the mean of four replicates 士 SD and are

expressed per dry weight of fruit body. The lowest mean zinc concentration in V.

volvacea caps was 96.3ppm and the highest llO.Sppm. Zinc concentrations in the

caps were virtually constant regardless of zinc concentrations present in the

substrate (Figure l).The lowest mean zinc concentration in V.volvacea stalks was

Sl . lppm and the highest 77.5ppm. Zinc concentrations in the stalks also varied

very little regardless of zinc concentrations in the substrate (Figure 2).

3.1.2. Uptake of Zinc by P. sajor-caju.

Figures 4-6 show the mean zinc concentrations in cotton waste substrate and fruit

bodies harvested after 28 days. Values are the mean of four replicates 土 SD and

are expressed per dry weight of fruit body. The lowest mean zinc concentration in

P.sajor-caju caps was 76.3ppm and the highest 96.5ppm. Zinc concentrations in

the caps was virtually constant regardless of zinc concentrations present in the

substrate (Figure 4).The lowest mean zinc concentration in P.sajor-caju stalks was

61.8ppm and the highest 79.3ppin. Zinc concentrations in the stalks also varied

very little regardless of zinc concentrations in the substrate (Figure 5).

33

3.1.3. Uptake of Zinc by Ledodes. t i..

Figures 7-9 show the mean zinc concentrations in sawdu拜 substrate and fruit

bodies harvested after 68 days. Values are the mean of five replicates 士 SD and are

expressed per dry weight of fruit body. The lowest mean zinc concentration in »

Ledodes caps was 132.5ppm and the highest 456ppm. Zinc concentrations in the

caps were virtually constant regardless of zinc concentrations present in the

substrate when zinc was added at levels between 0 and 400ppm. When zinc was

added to the substrate at levels between 600 and SOOppm the zinc concentrations

were slightly higher in the caps compared to those from lower zinc treatments.

(Figure 7). The highest zinc level in the caps was found when zinc was added to

the substrate at a concentration of lOOOppm. When dry weight zinc concentrations

of the caps and the lOOOppm treated substrate were compared the zinc

concentration in the caps were substantially lower than that of the substrate. The

lowest mean zinc concentration in L, edodes stalks was 120ppm and the highest

1621ppm. Zinc concentrations in the stalks increased with increasing zinc

concentrations which were present in the substrate but the absolute level of zinc in

the stalks did not reflect the level in the substrate (Figure 8).

34

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Added Zinc (g)

r ig 1. Mean zinc concentrations in cotton waste substrate

and V. volvacea caps. Values represent the mean of four

replicates. Error bars represent the standard deviation.

35

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Fig 2. Mean zinc concentrations in cotton waste substrate

and K volvacea stalks. Values represent the mean of four

r ；plicates. Error bars represent the standard deviation.

36

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Added Zinc (g)

Fig 4. Mean zinc concentrations in cotton waste substrate

and P. sajor-caju caps. Values represent the mean of five

replicates. Error bars represent the standard deviation.

38

3000 1 , r——I 1 1 1 1

g 2700 - • S t a l k s . N -

•rH kXNSubs t r a te v

d ^ 2400 - S -

fl 2 1 0 0 - S -QJ CD ^ \

吕 ^ 1 8 0 0 - N ^ -

O V \

^ fi 1500 - ^ ^ -

O ^ \ •S \ 1 2 0 0 - N ^ -

N 3 T ：\ ^ 900 - M \ ^ -

^ V N V

0) o \ o S 6 0 0 - O \ O -

0 r ^ r- iS l r 4 M r 4 o H N r l M _

0 0 .1 0 . 2 0 . 4 0 . 8 1.6 2 .0

Added Zinc (g)

Fig 5 Mean zinc concentrations in cotton waste substrate

and P. sajor-caju stalks. Values represent the mean of five

replicates. Error bars represent the standard deviation.

39

120

[ ]Stalks ~ bS'gcaps o 100 .~

+J ~ cd -+-l ~ ,.0

+J b.O ~.~ 80 QJ Q.)

(J ~ ~ >.

25 e5 60

(J OD ~, .~

N ~ 40 ~'-'" cd QJ ~ 20

()

o 0.1 0.2 0.4 0.8 1.6 2.0

Added Zinc (g)

Fig 6. Comparison between the cap and stalk zinc levels of P. sajor-caju grown on treated cotton waste. Values . . represent the mean of five replIcates. Error bars represent

stCLndard deviation.

40

2 8 0 0 r 1 1 1 1 1 1 1

2600 - T - •

2 4 0 0 - d l C a p s W _

k\N Substrate \ 2 2 0 0 - \ -^ \

2 2 0 0 0 - \ -0 i \

二 1 8 0 0 - N \ -<d ^ N N 1 S 1 6 0 0 - N ^ -

Pi lafl O； O O s -s 1400 - N ^ ^ -

r j 2 o o - ^ ^ ^ -u u T V V V

a Q 1000 - ^ ^ \ -

獻 - N ^ ^ N -如 \ V \ \

600 - \ \ \ ： " ^ 4 0 0 - $ 》 $ 「 专 -

r, r ^ r S r T R -

0 _ I U I kN I K1 I K1 I K1 M N I K^ I K1 1

0 0.16 0.32 0.65 1.30 1.95 2.60 3.25

Added Zinc (g)

Fig 7. Mean zinc concentrations in sawdust substrate and

Ledodes caps. Values represent the mean of five replicates.

Error bars represent the standard deviation.

41

2 8 0 0 1 , , , _ _ I ‘ I " I I I 1 1

2 6 0 0 - T _ . 丨 —

2400 - IStalk ^ _

白 2200 - 四 S u b s t r a t e ：\ _

S 2000 - ^ 一 (d T \

i 1800 - K] T \ 一

S S 1 6 0 0 - \ r ^ N -S 如 X \ N o '53 1400 - \ _ o ^ \ \ N

o 1 2 0 C - N ^ -

若占100。— S 广 $ 「 》 〉 -_ - ^ ^ ^ -

6 0 0 - R ^ ^ ^ -

側 - r - N \ ^ \ \ -

- U ^ -Q t r - i _ _ L - L j M M _ _ K 1___ IN__I I

0 0.16 0.32 0.65 1.30 1.95 2.60 3.25

Added Zinc (g)

Fig 8. Mean zinc concentrations in sawdust substrate and

L.edodes stalks. Values represent the mean of five replicates.

Error bars represent the standard deviation.

42

卜，

2 0 0 0 , i 1~~-n , 1 , , — — . •

1 8 0 0 — . 丁 - 「

leaps

160C - ^ 3 s t a l k s pi —

\ 1400 - T ^ -

fl N

1200 - 丄 〉 -g � J IN \

S 1 0 0 0 - N O ^ -

口 ^ \ O o

。•二 V \ \ d ^ 800 - T \ \ 0 -

A )》$ o ^ 60C - \ \ \ 0 -N 、 400 — A � ： ^ 〉 广 > -d a ^ N \ O 。吣 ^ \ t \ 0 ^ 2 0 C — r ^ r ^ 「^^ \ 〉 -

。 丄 — L i 」 — 0 0.16 0.32 0.65 1.30 1.95 2.60 3.25

Added Zinc (g)

Fig 9. Comparison between the cap and stalk zinc levels of

L. edodes grown on treated cotton waste. Values represent

the mean of five replicates. Error bars represent standard

deviation.

43

3.1.4. Symptom > of Zinc Toxicity in L edodes.

The first harvest of fruit bodies was taken after 68 days. All fruit bodies appeared

normal except those growing on lOOOppm zinc (Figure 10). The caps were

smaller, harder, rounder and paler than controls. The stalks were thinner, longer

and some were slightly twisted. The second harvest was taken after 73 days. The

fruit bodies taken from the SOOppm treatment were abnormally twisted (Figure 12)

compared with controls (Figure 11). Fruit bodies from lOOOppm zinc were badly

twisted and smaller (Figure 13). Table 1 shows the levels of zinc recorded in the

caps and stalks o仏 edodes fruit bodies relative to the zinc concentration present

in the substrate. Zinc concentrations within control fruit bodies after 73 days

growth were similar to those harvested after 68 days. Caps harvested from

lOOOppm treatment after 73 days contained more zinc than those harvested after

68 days (775ppm compared to 456ppm). The levels in the stalks were lower (973

compared to 1621ppm) Caps harvested from the SOOppm treatment after 73 days

growth :ontained more zinc than those harvested after 68 days growth.

44

* . . .

«

I

C-

Fig 10. Morphological differences of L.edodes fruit bodies

grown for 68 days on zinc treated and untreated substrate.

Note the twisted longer stalks and the smaller caps of the

zinc treated fruit bodies. From left to right: Control,(no

added zinc) replicate E; l’000ppm zinc, replicates ABCD.

45

N

Fig 11.Normal control L edodes fruit body formed after 73

days grown on substrate which was not treated with zinc.

H l l i I 丨 ) A 4 5 ‘ 6 7 8 <? 10 I 丨 I? I • . - • I

. I \ . .. \ • •• > if _ .尊 i、. 姓 • ... -lr • -I

Fig 12. Abnormal fruit body formation of Ledodes grown

for 73 days on substrate treated with SOOppm zinc. Note the

twisted stalk.

46

* . . .

I

I

f ‘

國 W n • ， … s u 丄 丄 l 』 ] | J

Fig 13. Abnormal fruit body formation of Ledodes grown

for 73 days on substrate treated with lOOOppm zinc.

47

‘；..

！--

Zinc added (g) I

0 0.16 0.32 2.60 3.25 (

Cap 125 151 178 486 775

Stalk 110 114 150 ND 973

Table 1. The zinc content (|ig/g dry weight) in Ledodes caps and stalks grown on various

quantities of zinc. Fruit bodies were harvested after 73 days and analysed for zinc using

atomic absorption spectrophotometry. The basal level of zinc in the substrate was 43 |ig/g.

ND- not determined.

48

3.2 Growth Studies

3.2.1. Radial Growth Measurements .

Fungi were grown on agar containing different concentrations of zinc. The basal

level of zinc as determined by atomic absorption spectrophotometry was found to

be 2.3ppm.

Flammulina sp was the least tolerant of the flingi tested. This mushroom grew on

zinc concentrations ranging between zero and SOppm of added zinc. After 9 days,

radial grjwth was greatest in controls with no added zinc and decreased markedly

at added zinc concentrations above SOppm (Figure 14). P. sajor-caju grew on zinc

concentrations ranging from zero to lOOppm of added zinc. After 9 days radial

growth was greatest in controls with and decreased markedly at added zinc

concentrations above lOppm (Figure 15). Tricholoma sp grew on zinc

concentrations ranging between zero and 200ppm added zinc. After 13 days, radial

growth was greatest in controls and decreased markedly at added zinc

concentrations above lOOppm (Figure 16). Ganoderma sp grew on zinc

concentrations ranging between zero and 200ppm of added zinc. After 17 days

radial giowth was greatest in controls and decreased markedly at added zinc

concentrations above SOppm (Figure 17). L. edodes grew on zinc concentrations

ranging between zero and 200ppm. After 17 days radial growth was slightly

greater al added zinc concentrations of 0.1 and 5ppm. Growth decreased markedly

at zinc concentrations above lOppm (Figure 18). V. volvacea was the most tolerant

fungus tested. This mushroom grew on zinc concentrations ranging between zero

and lOOOppm and growth on 2000ppm began after 4 days. After 3.5 days radial

growth was greatest in controls with no added zinc and decreased markedly at

added zinc concentrations above lOOppm (Figure 19)

49

° ‘ — “ — ‘ — 1 — 1 — — 1 — — 1 — — , — — — —

1 一 ^ 一

0 ！ 1 1 1 ' 1 1 1 1

0 1 2 ； 5 4 5 6 7 8 9 10

Days

r ig 14. Radial growth measurements of Flammulina sp

grown on different concentrations (ppm) of zinc. Values

represent the mean of three replicates. Error bars represent

the standard deviation. Basal zinc level was 2.3ppm.

Symbols refer to additional zinc; OOppm •O.lppm; VSppm;

V I Oppm; • 50ppm.

50

8 I I 1 1 1 1 1 1 1

-f / 一 丁 一

丨口： 0 T r I I I I I — I — 1 — I —

0 1 2 - 3 4 5 6 7 8 9 1 0 Days

Fig 15. Radial growth measurements of Pleurotus sajor-

caju grown on different concentrations (ppm) of zinc.

Values represent the mean of three replicates. Error bars

represent the standard deviation. Basal zinc level was

2 3ppm. Symbols refer to additional zinc;OOppm •O.lppm;

•5ppm; V10ppm;n 50ppm;«100ppm;

51

g ‘ ‘ r — ~ I 1 ！ 1——

a - T

0 I i L _ I I 1 I I

0 2 4 6 8 10 12 14 16 e

Days

Fig 16. Radial growth measurements of Tricholoma sp

grown on different concentrations (ppm) of zinc. Values

represent the mean of three replicates. Error bars represent

tiie standard deviation. Basal zinc level was 2.3ppm.

Symbols refer to additional zinc; OOppm •O.lppm; VSppm;

VlOppm; • SOppm; • ! OOppm; A200ppm;

52

J

1 0 i 1 1 1 1 1 1 r — 1

g - /kl^ '

0 2 4 6 8 10 12 14 16 18 20

Days

Fig 17. Radial growth measurements of Ganoderma sp

grown on different concentrations (ppm) of zinc. Values

represent the mean of three replicates. Error bars represent

the standard deviation. Basal zinc level was 2.3ppm.

Symbols refer to additional zinc; •Oppm; OlOppm; V5ppm;

•50ppm; •lOOppm; A200ppm.

53

1 0 1 1 i 1 1 1 1 i 1 ~

9 - -

8 - -

„

0 2 4 . 6 8 1 0 1 2 14 16 18

Days

Fig 18. Radial growth measurements of Lentinus edodes

grown on different concentrations (ppm) of zinc. Values

represent the mean of three replicates. Error bars represent

the standard deviation. Basal zinc level was 2.3ppm.

Symbols refer to additional zinc; O0ppm;V0.1ppm; •5ppm

V10ppm;n50ppm; •lOOppm; A200ppm.

54

9 , 1 i

a - -“ 0 . 1

7 - 广 0 -

i I j。： ‘ 丨 ‘

0 1 2 3 f

Days

Fig 19. Radial growth measurements of Volvariella

volvacea grown on different concentrations (ppm) of zinc.

Values represent the mean of three replicates. Error bars

represent the standard deviation. Basal zinc level was

2.3ppm. Symbols refer to additional zinc; DOppm; •

0 1 ppm; VSppm; VIOppm; OSOppm; •!OOppm; A200ppm;

A500ppm;01000ppm.

55

3.2.2. Biomass Measurements.

The biomass of F /侧麵 / / " “ sp was slightly higher at zero ppm zinc but was not

significantly different to 0.1 ppm. After 9 days biomass was greatest in controls

and at O.lppm. Biomass decreased steadily at zinc concentrations above 5ppm

(Figure 20). After 10 days P. sajor-caju biomass was greatest at 0.1 ppm zinc

compared to other treatments. Biomass decreased at zinc concentrations above

and below O.lppm (Figure 21). After 12 days Tricholoma sp biomass was greatest

in controls and treatments between 0.1 and 5ppm. Biomass decreased markedly at

zinc concentrations above 5ppm (Figure 22). After 17 days Ganoderma sp

biomass was greatest at a zinc concentration of lOppm. Biomass decreased

markedly at zinc concentrations above 50ppm (Figure 23). After 17 days L. edodes

biomass was greatest in controls and treatments between 0.1 and 5ppm. Biomass

decreased at zinc concentrations above lOppm (Figure24). After 4 days

V.volvacea biomass was greatest at a zinc concentration of O.lppm. Biomass

decreased at zinc concentrations above and below O.lppm (Figure 25).

56

20 i i ‘ I I _ I I I

1 8 - . -

16 - 了 一

£ c e s I P a. £ a G. g-X (X cu a. ou ^ §： — a CI O o O d 们 — 们 2

Fig 20. Dry weight of Flammulina sp grown for 9 days on

different concentrations of zinc. Values represent the mean

of three replicates. Error bars represent standard deviation.

57

—I

_

_

1

1

1

_

1

‘

te

日IS §

g

„

I

.11!

red

- I^i

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0

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20 - 丄 T .

‘："IliiiM" g a G B B

S ' a P 5 S a cu 5 S! 5 ^ ^ a* A 斤 二 O4 o o § o o o lO — 10 — CV2

r ig 22. Dry weight of Tricholoma sp grown for 12 days on

different concentrations of zinc. Values represent the mean

of three replicates. Error bars represent standard deviation.

59

4 0 i ‘ ‘ ‘ ‘ ： i i

3 5 - _

3 0 - . - J - ^ 一

S I' • 6 a I I « g： 6 a a a ^ ^ 3 cu ou a. o 2 斤 二 a o o o o o o m ^ in w oi

Fig 23. Dry weight of Ganoderma sp grown for 17 days on

oifFerent concentrations of zinc. Values represent the mean

cf three replicates. Error bars represent standard deviation.

60

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ii

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s

3.2.3 Morphological Alterations due to Zinc Observed with Light and Electron

Microscopy.

Figure 26 shows the control hyphae (grown on agar medium with a basal zinc level

of 2.3ppm) oiFlammidina sp which are long and growing close to the agar. After

7 days growth on 50 ppm zinc the hyphae are shorter, highly branched and exhibit

a tendency to group together in clusters and rise up above the agar (Figure 27).

After 11 days growth on 1 OOppm zinc, Flammulina sp did not grow radially across

the agar but produced aerial hyphae and secreted a yellow compound (Figure 28).

P.sajor-caju exhibited a very distinctive mycelial morphology when zinc

concentrations in the growth medium exceeded 50 ppm. The hyphae branched

together in an irregular manner and clumps appeared at the edges of the branches

(Figure 29). Figure 31 is the control which shows the individual hyphae

independently spaced. Figure 30 shows one of the branches with the hyphae

originating from a localised area and branching outwards from a common origin.

Tricholoma sp exhibited similar mycelial morphologies to the controls at zinc

concentrations up to 200ppm. However, at concentrations above 200ppm more

aerial hyphae were evident (Figure 32). Figure 34 shows the control hyphae after

10 days. Dense bodies resembling chlamydospores, formed after 9 days in cultures

containing〉140ppm zinc. (Figure 33).

Under low zinc conditions, Ganoderma sp produced many strong filamentous

hyphae, on the suri^ce of the agar, and these outwardly branching hyphae were

less evident with higher zinc levels. Hyphae appeared more robust when the

treated flmgus was grown with 1 OOppm zinc.

63

Morphologically, L. edodes exhibited similar colonial morphology to controls

when grown in the presence of zin~ levels up to 50ppm. At this concentration the

mycelial Inargin became more defined. Controls had thinner wispy hyphae than

those at 100ppm which were thicker and more robust.

Figure 3 5 shows the growth of V. volvacea growing on different concentrations of

zinc. Growth on 2000ppm began after 4 days. At 1000ppm the mycelial mat of V.

volvacea became embedded in the agar. As this mycelium became older, the

mucilagenous morphology became more obvious (Figure 36). Figure 37 shows the

long, robust hyphae from a 4 day-old V. volvacea control with a few short stubby

hyphae. After 4 days growth on 2000ppm the short stubby hyphae become

predominant and the tips protrude from the agar (Figure 38). This morphology

could al~,o be seen at 1000ppm. Electron micrographs revealed that hyphae of

V. volvacea grown on 1000ppm zinc had a 'wrinkled' convoluted surface

(Figure39) while the surface of control hyphae had a smooth appearence (Figure

40).

64

I • '%X \ • . 、•

Fig 26. Normal hyphae of Flammulina sp (xl6) control,

grown for 7 days. Basal zinc level was 2.3ppm.

. 1

• .- — - -

’ - - — ‘ — — - - — = -

Fig 27. Clustering of Flammulina sp hyphae (xl6) grown on

SOppm zinc for 7 days.

65

I'

H E • • B U k C : ’ ' ;

f ig 28. Production of a yellow compound by Flammulina

sp after 11 days incubation on lOOppm zinc. Left,100ppm;

Right control (Basal zinc was 2.3ppm).

Fig 29. Hyphal branching of P. sajor-caju grown for 10

days on zinc treated medium. Upper left lOOppm; bottom

control (Basal zinc was 2.3ppm); Upper right SOppm.

66

_ U b i t t i i M M l ^ r i i i i i i l i g i i l M d f l l H H I H H

Fig 30.Independently spaced hyphae of P. sajor-cajii (xl6)

control, grown for 10 days.(Basal zinc was 2.3ppm).

— ….-——-——-、 …

/

r . . • . •

：丨� , ./

、 《 ：

Fig 31. Branching of P. sajor-caju hyphae (xl6) grown on

lOOppm zinc for 10 days.

67

4

I

Fig 32. Tricholoma sp grown for 14 days on zinc treated

medium. Upper left control (Basal zinc was 2.3ppm); bottom

200ppm Aerial hyphae were produced; Upper right lOOppm.

68

爾 ~ — - - - — — - • • . . - 一

一 - - - ^ - — - —_ - ..、... “

Fig 33. Tricholoma sp (xl6) grown for 10 days on 140ppm

zinc. Arrows indicate dense bodies similar to

ehlamydospores.

I

Fig 34. Tricholoma sp (xl6) control grown for 10

days.(Basal zinc was 2.3ppm)

69

隨丨 Fig 35. V. volvacea grown on zinc treated medium for 12

days. Left 2000ppm; top control (Basal zinc was 2.3ppm);

bottom lOOOppm; right SOOppm.

i M l L -- -- -- —— - _ — _ —

Fig 36. The mucilagenous morphology of V.volvacea grown

on lOOOppm zinc for 16 days. Hyphae were embedded in

the agar.

70

m Fig 37. V.volvacea (xl6) control grown for 4 days.(Basal

zinc was 2.3ppm). Arrows show the few short stubby

hyphae.

I

_

Fig 38. Short stubby hyphae of V.volvacea (xl6) produced

after they were grown on 2000ppm zinc for 4 days.

71

m Fig 39. Scanning electron micrograph (xlOOO) of the

amorphous surface of V.volvacea grown for 12 days on

lOOOppm zinc.

f ig 40. Scanning electron micrograph of V.volvacea control

(kIOOO) after 12 days growth in chelated liquid medium.

72

3.3 V.volvacea Staining Studies

3.3.1 Protein Staining of V.volvacea using Coomassie Blue.

Figure 42 shows the control, the agar in which contained a basal level of 2.3ppm

zinc. Figure 41 shows V.volvacea hyphae grown on medium supplemented with

lOOOppm zinc. Hyphal proteins were stained with Coomassie Blue after 4 days.

As can be seen from figure 41 darker areas stained around the tips of the hyphae.

An extracellular protein matrix was evident along the length of the hyphae and

especially around the tips. As the extracellular protein matrix is only present in

those hyphae grown on agar which had been treated with lOOOppm zinc, it is

possible that zinc induces matrix production. There was no extracellular staining of

the control hyphae (Figure 42).

73

i.

nHOB I cSEP^ I m i i B U M H i m i ^ i i i i i l i f c r 警 i i Fig 41. Extracellular matrix produced by V.volvacea (xlOO)

after 4 days growth on lOOOppm zinc. Hyphae were stained

with Coomassie Blue.

If?—"~— _• -—•“—• — - • - ‘ « ——- -. -it 1.，，. j

f .. i

Fig 42. V.volvacea control (grown on 2.3 ppm zinc).

Hyphae were stained with Coomassie Blue after 4 days

growth.

74

3.3.2. Zinc Staining by Dithizone and Fluorescence Staining by DAPI

O f all the trace elements stained in vitro with dithizone, zinc sulphate was the only

metal to produce a distinctive red colour (Figure 43). In vivo studies showed the

control hyphae, growing on 2.3 ppm zinc contained very little red stain. There

were a few localised areas around the cell wall that stained pink/red (Figure 44).

Hyphae which had grown on SOppm zinc contained numerous small localized areas

of red stain (Figure 45). V.volvacea grown on 100 to 500 ppm zinc contained

regularly spaced compartmentalised areas of red stain which were larger than those

seen at SOppm. The size of the localised area increased with increasing zinc

(Figures 48 and 49).

After staining with DAPI and dithizone it was possible to find hyphae stained

positively for zinc in areas which stained negatively for nuclei (Figures 48 and 49)

and negatively for zinc in areas which stained positively for nuclei (Figures 50 and

51).

75

}

., . . . . ：、

Fig 43. Characteristic colours of trace elements after in

vitro staining with dithizone. From left to right. Control

(stain only); CoS04； NaCl; KH2PO4; H3BO3； CaCb;

MnS04； FeS04； MgSCM; CuS04； ZnS04； Control (stain

only).

76

"Jr . ： ：

Fig 44. Hyphal staining of V. volvacea control (on a basal

zinc level 2.3 ppm) using dithizone. Mycelium (xlOO) was 6

days old.

i

， y ^ 、产�令

rL t^v . 為 ； -

Fig 45.Hyphal staining of V. volvacea grown for 6 days on

50ppm zinc. Mycelium (xlOO) was stained with dithizone

resulting in small localized areas of red stain.

77

m. .. V --—— I

I - 、：V • J 9 德 I

I 、 I \ I ^ 、j .

； 。 系 , i - • •

� ‘

*

Fig 46.Hyphal staining of V. volvacea grown for 6 days on

200ppm zinc. Mycelium (x 80 ) was stained with dithizone,

resulting in small regularly spaced localized areas of red

stain.

‘•： 、、 ‘ Vv

fc • • , f -

Fig 47. Hyphal staining of V. volvacea grown for 6 days on

SOOppm zinc. Mycelium (xlOO) was stained with dithizone

resulting in regularly spaced localized areas of red stain.

78

few, “

f A A 对 久 、 . . ， 、

Fig 48. Six day old V. volvacea grown on SOOppm zinc and

Viewed using light microscopy. Mycelium (xlOO) was

stained with dithizone and DAPI. Arrows show localized

areas within the hyphae which stained positively for zinc.

B^BPiB JI F,g 49. Six day old V, volvacea grown on SOOppm zinc and

viewed using fluorescence microscopy. Mycelium (xlOO)

was stained with dithizone and DAPI. Arrows show

localized areas within the hyphae which stained negatively

for nuclei.

79

I 較 . , 囊 - ’ 樣 r : •

會 …：^ • - - J ..， . 厂 . . 一 -.. . . ' . • 1 • . - V

‘ ^ * - ‘ f ‘‘ - , •

f ^ : . 、”錢 /-•• ： 1 - 、 1 實 J 广 M ^ ； ,f j| � ^ ^ , 1 、乂 ^

Fig 50. Six day old V. volvacea grown on SOOppm zinc and

viewed using light microscopy. Mycelium (xlOO) was

stained with dithizone and DAPI. Arrows show localized

areas within the hyphae which stained negatively for zinc.

F — 禪 ： 義 诱 i ^ ^ ^ j ； ^ ! ^ ’ 一. ， - . 八 - . J f , t

— % 、 ？ i

Fig 51.Six day old V.volvacea grown on SOOppm zinc and

viewed using fluorescence microscopy. Mycelium (xlOO)

was stained with dithizone and DAPI. Arrows show

localized areas within the hyphae which stained positively

for nuclei.

80

3.4. V.volvacea Protein Profile Comparisons after Gel Electrophoresis

Figure 52 illustrates the protein profiles of V.volvacea grown on different

concentrations of zinc ranging from O-SOppm. Mycelia were harvested after 4 days

growth，protein was extracted from the mycelial fractions and the samples

subjected to SDS gel electrophoresis. Overall the profiles obtained were similar

regardless of the zinc concentrations present in the medium. However, upon closer

examina tion the protein profile obtained from cultures grown in the absence of zinc

contained an extra protein band. This protein band could represent a zinc binding

protein which is produced in response to suboptimal zinc concentrations.

81

i • I

I ‘ ‘ ！. 一

I i ！

！ I

I ‘ -

‘, 丨 - 、 . 、― ‘

j 遍 ' m m .、.城. S^^mKt nafcii ^ .^B^MF

i 糖 画 ^ ^ H H P

； 完 费

m. £l L ― …

Fig 52. Protein profiles of V. volvacea grown for 4 days on

different concentrations of zinc. From left to right: Lane 1:

50ppm. Lane 2: 20ppm. Lane 3: lOppm. Lane 4: Oppm.

Arrow shows an extra protein band. 18 |ag protein was

added to lane 2 and lane 4. The gel was run at 240 volts.

82

4. Discussion

4.1 Z inc uptake by Frui t Bodies

4.1.1 Uptake of Zinc by V. volvacea and P, sajor-caju Fruit Bodies.

Mushrooms were grown on the conventional substrates to produce the maximum

number of fruit bodies. When cotton waste substrate was prepared, 2% lime was

added to increase the pH. These conditions are required for optimum fungal

growth. An acidic pH will free cations making them more availiable whereas

cation precipitation resulting in decreased bioavailability occurs at a higher pH.

Zinc forms a zincate ion which can be precipitated by calcium ions above pH 8.5

Ehrlich ；1971). Lime is known to reduce the bioavailiability of zinc, and zinc

deficient plants can be found in ecosystems which contain it. The deficient plants

are often those which do not have a mycorrhizal associate. Mycorrhizal fungi are

known to be able to mobilise metals, especially those needed for normal cellular

functioning, and pass them to the plant.

In this experiment V. volvacea and P.sajor-caju were tested for their ability to take

up zinc. This may occur either passively by adsorption or actively using a

metabolism-dependent uptake mechanism whereby hyphal secretions may mobilise

zinc and facilitate its absorption into the fruit bodies. Figures 1,2,4 and 5 show zinc

levels in the caps and stalks of V. volvacea and P. sajor-caju remained constantly

low. Th丨：；ability to take up the metal under conditions when zinc availiability is

restricted would render the flingi useful for bioremediation programmes for

stringent conditions where zinc is in an unavailable form in the environment.

It is possible that the zinc was in an available state but was not taken up by fruit

bodies. There may be two mechanisms which kept zinc levels constant. One

enabled them to accumulate zinc to the required quantity when substrate levels

83

t

were lov.'er than the mushroom zinc requirement level. The other came into effect

under high zinc concentrations and is a preventative zinc uptake mechanism. The

purpose of this mechanism is to ensure that zinc is not taken up in quantities that

may be toxic. Some mycorrhizal fungi are able to load their mycelium and protect

their plant hosts ( Gast et al, 1988). The mycelial zinc loading capacity is probably

related to the amount of mycelial biomass and the more biomass, the more efficient

it is at preventing zinc transportation to the fruiting bodies. The zinc loading

capacity theory probably has limitations which would be exceeded beyond the

range of zinc used in this experiment.

It was interesting to note that the caps contained more zinc than the stalks.

(Figures 3 and 6). This probably reflects the need for zinc as a micronutrient in the

caps. The caps may act as a sink because this area is more active in cell division

than the stalk tissue.

4.1.2. Accumulation of Zinc by L edodes Fruit Bodies and Mechanism of Toxicity.

Both the control and treatment 2 of L edodes caps and stalks accumulated zinc to

a constant level (Figures 7 and 8). This is probably the optimum zinc level required

for norn.al growth and at these low levels of zinc treatment the mushroom may

have a mechanism which keeps its internal levels constant.

When compared to the control, zinc levels within the mushroom caps of treatment

3 and treatment 7 increased to a constant level, and in treatment 8 they were

higher (Figure?). Morphological differences could be seen when the fruit bodies in

84

this treatment were compared to the control (Figure 10). The caps had a smaller

diameter, were rounder, lighter and hard to the touch.

Generally the stalks accumulated more zinc than the caps (Figure 9). The zinc level

in the caps may be lower because the fruit bodies were harvested as soon as they

matured and this may not have given the fungus enough time to transport the zinc

from the stalks into the caps. Stalks accumulated zinc beyond treatment 3 and the

level of zinc increased with an increase in substrate concentration. A few of the

stalks were slightly twisted in treatment 8 (Figure 10)

The second harvest , taken 5 days after the first produced fruit bodies which had

similar quantities of zinc in the treatment 1,2, and 3. Treatment 7 produced fruit

bodies with much higher levels of zinc in the caps (Table 1. Figures 11 and 12).

There are abnormal distortions of the caps and twisting of the stalks. Treatment 8

producec fruit bodies with higher zinc levels than those of the same treatment

taken from harvest 1. These abnormals were very weakly, small, twisted and

stunted (Figure 13). The elevated levels of zinc found in the caps of harvest 2

compared to harvest 1 could be due to zinc accumulation over the 5 extra days

that the mushroom took to mature or it could be due to the lack of watering that

was administered during these 5 days. It would be interesting to see the levels of

zinc in fruit bodies which were not watered over the whole growth period.

The toxic effects of zinc on L edodes caused the fruit bodies to become smaller,

abnormally twisted, harder and lighter (Figure 10). Similar toxicity symptoms

have been described by Lambinon et al (1964) in Cladoma cornntoradiata and C.

pyxidata. These lichens formed small podetia which were granular squamous and

twisted.

85

The abnormal morphologies could be due to a number of mechanisms, the most

well documented being the disrupted transport of potassium, sodium, chloride ions

or organic molecules across membranes and the poisoning of enzymes. The

binding of zinc with membranes is thought to affect the permeability. This has been

well documented in plants such as Agrostis tenuis, which is tolerant of many

metals. Wyn Jones and Sutcliffe (1972) reported zinc to cause a greater leakage of

potassium from non-tolerant rather than tolerant grass.

The most likely mechanism leading to abnormal growth is the disruption of normal

enzymic activity. Cell elongation depends partly upon the synthesis of cell wall

material and irregular synthesis may cause the abnormal twisting of the stalks

during development and also the malformation of caps. The disruption of enzymes

involved in the synthesis of polysaccharides could cause severe malformations in

fungi because of the numerous carbohydrate derivatives which make up the cell

wall. Woolhouse (1970) proposed p-nitrophenylphosphatases were involved in

polysaccharide synthesis in plants and Cox and Thurman (1978) went on to find

zinc inhi^^ited cell wall acid phosphatases of Anthoxanlhum odoratum. Wainwright

and Woolhouse (1978) found the same inhibition in Agrostis tenuis.

Zineb is a zinc based multisite fungicide which is a non-specific inhibitor of

respiration. It's effectiveness against respiration is because it reacts with

sulphydryl-group containing enzymes. Aconitase, an enzyme associated with the

tricarboxylic acid cycle is inhibited by ziram, a zinc based fungicide. Ziram is a

fungitoxic chelating agent that exchanges zinc for the ferric ion of aconitase Owens

(1960). Zinc therefore can replace structurally important elements within the cell.

Further studies should be carried out to find out the significance of decreased

86

enzyme activity, decreased respiration and increased membrane permeability with

morphological alterations.

The zinc levels in the controls of the fruit bodies of the three mushroom species

varied. F. sajor-caju contained approximately 70 |.ig/g dry weight. V. volvacea

contained approx 100 ng/g (Figure 1 and Figure 4). These controls were grown

on the same substrate which indicates variation is species specific rather than

substrate specific. Reports of Ledodes zinc levels differ between various groups

and this has been attributed to the different substrates used by different workers.

The properties of L edodes as an aphrodisiac have been attributed to high zinc

levels in fruit bodies (Flynn, 1991). In this experiment the zinc level in the caps was

approx i30ppm which is higher than both V. volvacea and P. sajor-caju. The

difference may be due to the species or to increased bioavailiability of zinc in the

different substrates.

87

4.2 Effects of Z inc on Growth

Each of the six mushroom fungi exhibited reduced growth rates in media

supplemented with zinc concentrations of SOppm or above (Figures 14-19). Zinc

most probably reduces the growth by interfering with normal cellular functioning,

possibly by blocking cellular reactions and metabolic pathways. It is feasible that

under conditions of environmental stress fungi will use alternative pathways in

order to survive. One of the fungi investigated, Flanwuilina sp, released a yellow

compound into medium treated with 1 OOppm zinc (Figure 28).This may be used

as a chelating agent which is formed in a final attempt to detoxify its surroundings.

Fungi are known to deposit yellow hydrogen sulphide around the mycelium under

conditions of stress (Gadd and Griffiths, 1978).

The hyphae of Flcwmnilina, clustered together in the presence of zinc. Both P.

sajor-caju and Ganoderma sp produced many branching hyphae from the same

hyphal ciigins at the edge of the mycelium when treated with zinc (Figure 31). The

hyphae of Tricholomci sp were primarily aerial at 200ppm (Figure 32). It is

possible chat for an individual hypha, the degree of exposure to zinc is lower in a

dense mycelium (Darlington and Rauser, 1988). This is probably why the

mycelium clumped together either in clusters or in branches. For L edodes the

hyphae were thicker and fewer when treated with zinc. Fewer hyphae, which were

embeddtd in the agar led to more contact with zinc but a wider thicker hypha with

a larger surface to volume ratio would not internally be exposed to as much metal

as a small surface to volume ratio.

88

Figures 20-25 illustrate that an increase in zinc concentrations in the culture

medium causes a decrease in fungal biomass. Colpaert et al (1992) suggested that

the more fungal biomass, the more zinc that can be immobilised. In mycorrhizal

flmgi it may be that plant tolerance in zinc contaminated soils is due to the fungal

associate being able to produce substantial amounts of fungal tissue following

metal exposure. Colpaert et al. (1992) found that plants with a sparse mycelial

associate contained higher zinc levels. Accumulation was lower in plants which

were infected by fungi with a dense mycelium. It may follow that the zinc retention

capacity of a limited mycelium is more restricted.

Possible mechanisms of resistance to metals may depend upon intrinsic properties

or genetic or physiological adaptations. Ashida (1965) has summarised

mechanisms of resistance as follows. The fungus may be able to bypass an inhibited

reaction or increase the concentration of the enzyme that the metal inhibits. It may

have a decreased requirement for a product of an inhibited metabolic system. The

concentration of a metabolite that antagonises the inhibitor may be increased or

there may be an increase in the production of a substance that makes the inhibitor

inocuous by binding it. Finally there may be physiological alterations leading to

decreased permeability of the cell to the inhibitor.

4.3 V.volvacea Mechanisms of Tolerance

Some fungal species are able to overcome the toxic effects of metals and there are

numerous ways they can do this. In this experiment V. volvacea appeared

mucilagenous (Figures 29 and 30). It has been reported that slime moulds are very

resistant to the toxic effects of zinc (Setala and Nuorteva, 1989). Zinc in Fuligo

septica was 4,000-20,OOOppm. The characteristic thought to play a role in

tolerance is the slime barrier. After staining the hyphae of V.volvacea at 1,OOOppm

89 I'

with Coomassie Blue an extracellular protein matrix was seen (Figures 40 and 41).

The presence of zinc triggers the production of the extracellular protein which may

have a protective role that prevents the uptake of zinc by the flmgal hypha. It is

unlilcely that the zinc is taken up by the hypha and then deposited in the matrix

because generally, resistant strains offlingi take up less metal (Gadd，1986). I f the

zinc has been taken up intracellularly, compartmentalised and transported within

the hypha then there may be a mechanism by which the zinc is excreted from the

hyphal tip. The Coomassie Blue shows the protein to be primarily located around

the tip and this was thought to be a zinc binding protein that is released into the

medium The matrix might therefore serve to absorb or be involved in the

precipitation of metals around the hyphal surface. Dithizone, which stains zinc a

characteristic red colour, was used to further this study, but other techniques such

as radiolabelling of zinc and transmission electron microscopy could also be used.

Dithizone is a very specific stain which is extremely sensitive and it was important

to prevent zinc in the agar from touching the mycelium as this could lead to false

positive zinc detection. The use of a coverslip provided the best way of preparing

hyphal tips which contained no surface zinc. It was difficult to prepare hyphae at

the lOOGppm zinc level because the mycelium was embedded in the agar and did

not grow up onto the coverslip. Using this technique any positive result could be

assumed to be due to the transportation of zinc from the mycelium toward the tip.

The localised red circular hyphal staining suggests the zinc is compartmentalised in

an organised manner along the hyphae. The function of this compartmentalisation

may be for the storage of zinc and the subsequent removal of it from the cell but

no zinc was detected beyond the hyphal tip.

90

Electron dense bodies which were thought to contain zinc have been recorded by

Paton and Budd (1972) in Neocosmospora crassa and copper is known to attach

to the nucleoli and chromosomes of yeast (Lindergren, 1971). In V.volvacea, the

even spacing with which the red stain appeared along the hyphae suggested the

zinc was associated with nuclei (Figures 46 and 47). It is possible that the zinc is

precipitated around the nuclei as part of a detoxification mechanism. Intracellular

precipitation, around nuclei and within vacuoles has been observed in algae

(Fernstenberg et al, 1975). It is possible that at higher zinc concentrations the zinc

is associated with nuclei and at lower zinc concentrations it is associated primarily

with vacuoles (Figure 45). This hypothesis however is contradictory to the

observations made by Ferstenberg et al. (1975) who found electron dense bodies

containing copper in the nucleus at low concentrations and in vacuoles and nuclei

at high concentrations.

Surprisingly, after staining with DAPI there was no correlation between the

nuclear staining and the presence of zinc (Figures 48-51). DAPI is known to stain

nuclei by binding to the AT rich double strand DNA and dithizone acts as a

chelator to form a zinc dithizone compound complex. It is possible that in older

hyphae partial nuclear degenerated may have occurred and this prevented DAPI

staining. The zinc may have accumulated around the nuclear area before

degeneration commenced and thus there was positive staining for zinc but negative

staining，br nuclear material. To account for positive nuclear staining and negative

zinc staining it is possible that zinc is not present in all nuclei. Nuclear localisation

of zinc may be dependent on the stage of nuclear division, its activity or the age.

Some fungi have a fungal sheath around their hyphae. If the protein matrix is a

fungal sheath it may have become thickened under conditions of zinc stress. It is

91

possible that the matrix visualised with Coomassie Blue, is a fungal sheath that acts

as a barrier rather than having a direct detoxification fianction. Scanning electron

microscopy of the control hyphae showed they had a smooth surface whereas the

zinc treated hyphae had an amorphous surface (Figures 39 and 40). The

amorphous surface could represent the same protein that was stained with

Coomassie Blue. The dithizone stain did not show zinc to be present outside the

hyphal tip so the function is probably to act as a zinc barrier.

Scanning electron microscopy of V. volvacea fruiting bodies and hyphae has been

carried out by Li (1982). The hymenial layer of a mature fruiting body has an

amorphous surface which is remarkably similar to the hyphal surface produced in

zinc treated conditions of lOOOppm. It is possible that the addition of zinc triggers

specific genes into the producing the amorphous surface all over the mycelium.

Zinc is known to play a role in reproduction and this has been linked to the zinc

containing enzymes. Perhaps there is another function that can be linked to

morphological changes associated with the hymenium.

More rosearch is needed to compare different fungi for the the presence of

structures such as the sheath and also components of the cell wall. The cell wall

may play an important role in metal binding and if the overall composition of

different fungi is not the same there could be a direct influence on binding.

Mann an s, glucans, phosphomannans, melanins and chitin and chitosan are all

possible agents to which zinc may bind (Gadd, 1991).

V. volvacea was the only fungal species with its mycelium embedded in the agar

under zinc conditions that slowed growth. When the base of the dish was viewed

using u e microscope the mycelium at lOOOppm had readily produced

92

chlamydospores. Chlamydospores, and the presence of melanin have been reported

to play a role in metal binding (Mowll and Gadd, 1984; Rizzo et a/.. 1992). They

may play a role in V.volvacea by accumulating the metal in their cell walls. The

micrograph of Tricholoma sp growing on 200ppm shows the presence of

chlamydospore-like structures (Figure 33). This fungus was the second most

tolerant of zinc but grew away from the zinc treated agar. The fungus avoided

growing on the agar by producing aerial mycelium. Tolerance may be a

consequence of one or numerous mechanisms acting at one time and the more

mechanisms the fungus has, the more metal it will tolerate. It is also possible that

one particular mechanism will be more effective than another. The ability of

V,volvacea to produce a protein matrix may for example have been more effective

than the production of chlamydospores. If fungi are more susceptible to metals

because their membranes become more permeable and increase the exposure of

metal binding sites then an efficient protective mechanism such as the presence of a

protein matrix would render a fungus more tolerant by protecting these binding

sites. V. volvacea could be substantially more tolerant (2000 opposed to 200)

because it has a more effective mechanism or because it has a combination of

mechanisms (extracellular protein barrier, chlamydospore production and possibly

other biosorption agents that are not as common in the other tested fungi.)

Tolerant fungi may have the ability to overproduce a factor which is involved in

metal detoxification. The metal itself would trigger the switch into overproduction.

Other fungi may be unable to overproduce possibly because the metal itself

interfere, with the metabolic pathway that leads to the formation of the protective

mechanism.

4.4 Differences in Protein Profiles of K volvacea Grown on Different

Concentrations of Z inc

93

In this experiment gel electrophoresis was used to examine the protein profiles of

V.volvacea. Figure 52 shows one protein band was present under conditions of

limited zinc and it was not present under supplemented conditions. This protein

band may be associated with the presence of a zinc scavenging protein such as a

siderophore. It may itself be a zinc binding protein or it could be an enzyme

associated with the production of a low molecular weight binding protein. Brot

and Goodwin (1968) found E.coli produced a red low molecular compound which

complexed inorganic iron to form a coloured iron chelate. This group went on to

discover the enzyme which catalysed the synthesis of the compound. Labelled zinc

could be used to investigate if the protein is a binding protein or an enzyme.

The addition of small amounts of a particular metal to the medium often has a

marked effect on the production of a certain metabolite. When zinc was added to a

growing culture of Ustilago sphaerogena、the amount of ferrichrome produced

decreased sharply (Neilands, 1953). In the present experiment, V.volvacea culture

may produce a metabolite in response to the limitation of another nutrient after

growing for a few days. But the presence of zinc in the supplemented medium may

cause a reduction in the biosynthesis of that metabolite and hence it is not present

under zinc supplemented conditions.

It is possible that the protein is a zinc binding protein and this could be tested using

an autoradiogram. If the protein has taken up zinc in the supplemented medium

then the uptake of zinc may have caused its molecular weight to become higher

and thus prevent it being in the same location in each of the lanes. This hypothesis

could be tested by running an autoradiogram and scanning the whole gel for zinc

binding proteins.

94

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