The association of higher fungi and sand dune grasses

THE ASSOCIATION OF HIGHER FUNGI AND

SAND-DUNE GRASSES

Ruby Ione McKay

A Thesis Submitted for the Degree of PhD

at the

University of St Andrews

1968

Full metadata for this item is available in

St Andrews Research Repository

at:

http://research-repository.st-andrews.ac.uk/

Please use this identifier to cite or link to this item:

http://hdl.handle.net/10023/14252

THE ASSOCIATION OP

HICmER RJNGI AND SAND-DUNE GLASSES.

by

Raby I . MoKay# B.So.

A th e s is subm itted to th e U n iv e rsity o f St# Andrews fo r th e degree o f D octor o f Philosophy#

ProQuest Number: 10166627

All rights reserved

INFORMATION TO ALL USERS

The quality of this reproduction is dependent upon the quality of the copy submitted.

In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed,

a note will indicate the deletion.

uest

ProQuest 10166627

Published by ProQuest LLO (2017). Copyright of the Dissertation is held by the Author.

All rights reserved.

This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO.

ProQuest LLO.

789 East Eisenhower Parkway P.Q. Box 1346

D E C L A R A T I O N .

I hereby d e c la re th a t th e follow ing T hesis is based on a record of work done by lae, th a t th e T hesis is my own com position, and th a t i t has not p rev io u sly been p resen ted f o r a H igher Degree.

C E R T I F I C A T B.

I c e r t if y th a t Ruby I . McKay has spent n ine term s of re se a rc h work under my d ire c tio n and th a t she has f u l f i l l e d th e co n d itio n s of Ordinance No* 16 (st*

C A R E E R .

In 1959f I was awarded a Jam aica Government

Teacher’ s S cholarship te n a b le a t th e U n iv ersity of th e West In d ie s, K ingston, Jam aica, from which I graduated in Ju ly , 19^3 w ith a f i r s t c la s s honours B achelor of Science (London) degree

in Botany and Zoology.

I tau g h t Botany and Biology in S t. Andrews High School f o r G irls in K ingston, Jam aica f o r two y e a rs.

I was then awarded th e S ir James Irv in e Memorial

S cholarship, te n a b le a t th e U n iv ersity of S t. Andrews, Scotland, I m a tric u la te d a t th e U n iv ersity of S t. Andrews

in O ctober, 19&5 and was adm itted as a Research Student under Ordinances l6 and 61.

A C K N O W L E D G E M E N T S .

I wish to express my thanks to r ro fe s s o r

J . A. Macdonalu, my «v.^crricor Tfho suggested the problem, f o r h is advice and in te r e s t during the course o f th is work.

I am also in d eb ted to th e S t a t i s t i c s Department o f thh U n iv e rsity o f S t. jlndrews f o r s t a t i s t i c a l l y

analysing th e d ata in S ections and o f th is th e s is . I would lik e to take the o p p o rtu n ity to e::prccL ray g ra titu d e also to th e U n iv e rsity o f the .e s t In d ies f o r awarding me the a i r James Irv in e Memorial S cholarship and to S ir H arold M itch ell Bt#, M.A.'j Hon. LX.D., K. S t. J . , who provided the funds, thus making i t p o ssib le fo r me

C O N T E N T S

Pag,

S ectio n 1# In tro d u c tio n •• . . #. 1 S ectio n 2. M a terials and General Methods • • • • • # 3

2a# M a te ria ls ... 3 2h# G eneral methods •• •• ». •• •• •• •• .# 6

2 h (l) S te r ili z a t io n ... .. •• . # # 6 \ a ) glassw are . . ••... 6 ,h) c u ltu re media « . . #. 6 c) caryopses #. . . •• •• •• •• •• 7 d) f r u c tif ic a tio n fo r is o la tio n #. . . 7 e) ro o ts f o r is o la tio n . . . . , . ## 7 ( f ) earthenw are p o ts and sand ... 8 2 b ( ii) Exam ination o f ro o ts f o r in fe c tio n #. 9 ro o ts from p la n ts in Tentsm uir . . .# 9 ro o ts from p la n ts in th e green house •• 9 2 b ( iii) fH m ^l is o la tio n s from ro o ts . . . . . . 10 2 b (iv ) F ungil is o la tio n s from f r u c tif ic a tio n s • 12 2b(v) Growth o f se e d lin g s • • • • • • • • • • 13 S ectio n 3# M orphological and c u ltu r a l c h a r a c te r is tic s

o f th e is o la te s #« . . •• •« •• •• 13 S e c tio n 4# P h y sio lo g ic a l c h a r a c te r is tic s o f th e

isolates ## #. ••

•• #. •• ## ##

21

4a* Vitamin requirmaent . . «• •• •• •• #,

•• 21

method .*

*. .* #. #. ## #* *. ••

21

results ##

. . •• •• •• #. .* •• ••

23

discussion •• •• «•

•• *. •• •• ••

27

4b# Utilization of nitrogen sources . . . . . . ##

30

method # »

#. . . #. ## •• •• ## ##

30

r e s u lts ## ## . . . . ... 32 d isc u ssio n #. #. .# . . .# #. e . ## 34 4c# P roduction o f enzymes •# ## 36 4 c (i) P e c tin o ly tic enzymes ## ## #. ## ## 36 method ## ## •• #. ## ## ## ## 36 r e s u lts •• .# #. #. 39 d isc u ssio n •• ## ## ## ,# ## ## 41 4 o ( ii) C e llu lo ly tic enzymes •« .# #* #. # 48

method ## ## ## ## 48

r e s u lts ## •• .# ## ## #, ## 30

d isc u ssio n # # # # # # # # # » # # # # 3^ 4 o ( iii) P roduction o f polyphenol oxidases ## ## 36

4d# D écom position o f n a tiv e lig n in and c e llu lo s e 60 method «• #. •• •• •• #* ». # # ## 6 l r e s u lts •• #. #. ## #. ## •• .# ## 6 l d isc u ssio n .# •• # * #. # *. ## #* 61 4e# Spore g ezn in atio n . . ## .# ## »# ## .# 63 Experim ent A #. •• #. •• .» •• #. 63 method •• •• . . .# *# •• •• •• 63 r e s u lt •• •• #. •• •• •• ## e# 63

Experim ent B * •• . . •• •• 63

method #. •• «• •• ## # ## 63

r e s u lt *. #. .# ## 63

Experiment C #. *. •• ## •« . . ## 63

method «• •• ## •• •• 63

result •• •• •• •• •• •• .# ## 63

Experiment D . . *. •# ## •• •• •• 66

method #. #. «• •• •• *# ## ## 66

resu lt . * , , # # ## # # ## •• •• 66

Experiment E ## •• . . •• . . .# •• 66

method ## •• •• •• •# •• •• ** 66

resu lt #. $. #. *# •• #. . . e 68

Discussion •• «• •• •• # # •• •* •• 68

S ectio n 3# In te ra c tio n s o f fungi and g rasses . . 71 3a. P rod u ctio n o f s y n th e tic mycorrhissa •• •• .# 71

3 a (i) Method ... 71 3 a ( ii) R esu lts and d isc u ssio n s ... 74

(a ) Sporophore p ro d u ctio n •• •• . . 74 r e s u lts . . •• •• •• #. 75 d isc u ssio n •• •• •• •• .# 76 (b ) Dry w e i ^ t s o f th e a e r i a l p a rts •• 83 r e s u lts •• •• •• . . •« •# 83

d isc u ssio n . . .# . . .# #. 87

(c ) Root in f e c tio n •• •• . . #. .# 90 r e s u lts . . .# #. •• .# 90

d isc u ssio n .# . . . . #* .# 95 3b. Incidence o f in fe c tio n in th e f ie ld . . . . IO4

3c. The e f f e c t o f ro o t e x tra c t a g a r on th e growth

o f th e is o la te s . . . . . . . . . . . . IO9 method •• •• •« «• . . . . . . «. .# 1(9

r e su lts •• •• •• •• •• •• •« . . .# 110

d iscu ssio n . . . . .# . . . . . . . . . . 113

3d. Production o f root exudates and th e ir e ffe c ts

on th e growth o f th e is o la te s . . . . . . . . 113 method #. . . .# #. . . . . . . . . . . 113 r e s u lts .# . . •• •• .# . . . . . . 118

S E C T I O N 1 INTRODUCTION

During re c e n t y e a rs th e b io lo g ic a l and chem ical compo­ s itio n o f sand-dunes has been th e su b je c t of f a i r l y exten­ s iv e in v e stig a tio n s# The c o n trib u tio n to th e Tcnowledge of th e m icro-organism s in th a t ec o lo g ic al n ich e is m ainly based upon th e voxk o f Moreau (1941, 1942), Vebley, Eastwood & Gimingham (1952), Brown (1956)# N icolson (1959, I9 6 0 ),

Pugh (1962) , Pugh, Blakesman, Morgan-Jones and Eggins ( I963) and Hassouna and Wareing ( I965)* That th e array of low er fungi in sand-dunes is v eiy wide was shown by Moreau

(1941, 42), Pugh (1962) and Pugh e t a l (1963). S o il fu n g i o f some B r itis h sand-dunes in r e la tio n to s o il ty p e and c o lo n isa tio n by h ig h e r p la n ts have been stu d ie d by Vebley e t a l (1952) and Brown (1956)# Hassouna and V areing

(1965) c o n trib u te d by d isc u ssin g th e p o s s ib le ro le o f rh is o - sphere b a c te r ia in th e n u tr itio n of Jbnmophila a re n a ria L# w hile N icolson*8 (1959,60) c o n trib u tio n was a study of th e v e s ic u la r-a rb u s o u la r ty p e of m ycorrhiza in g ra sse s confined to th is area.

Dobbs and Gash ( I965) have re p o rte d th e presence o f m icro b ial and re s id u a l m ycostasis in s o ils w ith low n u tr ie n t oonte n t e #g# sand-dunes# The n u tr ie n t s ta tu s o f th e

2

th e a re a reooivee sea sp ray s which c o n ta in a p p ré c ia b le q u a n titie s o f th e se ions# P otassium , n itro g e n and

phosphorus have been re p o rte d to be extrem ely low in q u a n tity in th e b are sand a s w ell a s in th e co lo n ized a re a s o f th e fo re dunes#

A lth o u ^ f x u it b o d ies o f th e h ig h e r fungi have been observed on th e sand-dunes sin c e th e l a s t ce n tu ry , very

l i t t l e is known about t h e i r n u tritio n # The purpose o f t h i s e x e rc ise i s , th e re fo r e , an attem p t to fin d out w hether th e re i s a n u tr itio n a l re la tio n s h ip e x is tin g between th e h ig h e r fungi and th e grasses# C e rta in ly , they show s p e c ia liz a tio n by growing only on th e dunes where p a r tic u la r g rasse s

predom inate, and th e r e g u la r ity w ith which they f r u i t each y e a r su g g ests th a t th e re i s a lin k between th e se two groups o f p lan ts#

This work was c a r r ie d out on th e fo re dunes (between 20 and 40 f e e t above h i ^ w ater mark) a t th e N ature Reserve in T entsm uir, F ife (P la te 1, Grid ref# 56/5026 06 map o f G reat B rita in )# In th is re g io n , c o a s ta l a c c re tio n i s proceeding mere ra p id ly th a n in alm ost any o th e r p a rt o f B rita in # This

ra p id f i n i n g o f ground from th e sea a ffo rd s ex c ep tio n al o p p o rtu n itie s f o r th e stu d y o f th e c o lo n iz a tio n by h ig h e r p la n ts and fungi# The th re e dominant sp e cie s o f h i ^ e r p la n ts which co lo n ize th e dunes a r e Agropvron .hinceifoxM A. & D, Leva, L. and Aamophlla a re n a ria L .. A. junceifoCT^e i s more abundant and A# a re n a ria le s s

abundant tow ards th e h i ^ w ater mark# The re v e rse is tr u e a s th e dune becomes more fixed# B. a re n a riu s occupies an

in term ed iate p o sitio n #

The f r u i t bodies o f th re e A garics and one Discomyoete occur in high frequency on th e dunes among th e g rasses

PLATE 1

m m m

I #

• '

3

th e nom enclature o f th e se fungi b u t a f t e r r e f e r r in g

to Saccardo (1888 and 1889), Lange (1936-40), N etrod (1941#

1942) , Andersson (l9 5 0 ), Dennis (196O), Orton (196O), D ennis, Orton and Hora (196O), S in g e r (1962) and W atling (p erso n al communication) th e fo llo w in g names have been a s c rib e d to th e re g u la rly o c c u rrin g sand-dune fU ngi, ( i ) Conocvbe dunensis. W allace 196O#

( i i ) M elanoleuca grammooodia (F rie s ex B u llia rd ) Pat# I9OO# Many accounts o f sand-dune fungi in c lu d in g th a t o f W allace

(1954) , r e f e r to th is fungus a s Trioholoma melaleuoum (Pers* ex Fr# 1921) Bummer I87I , but th e am yloid w arts o f th e spores and harpoon-shaped q y s tid ia keep i t in th e genus M elanoleaca

(O rton, i960 and S in g e r, I962)*

( i i i ) P s a th v re lla ammoohila (D urieu e t L e v illa ) Orton I96O# T his fungus has p re v io u sly been reco rd ed , f o r example, by Andersson (l9 5 0 ) and W allace (1934) &Q an o b lig a te sand-dune

fungus under th e name o f Psilocarbe ammouhila (D urieu e t L e v ille , I868) O ille t 1874# b u t because o f i t s d is tin c t c e llu la r e p ic u tis i t has been given th e newly combined name by Orton ( I96O)#

( iv ) P eaiaa ammoohila (D urieu e t Montagne) Cooke# This Discomyoete has been c a lle d Qeopyxis amroophila in many papers which d eal w ith sand-dune fungi e#g# W allace (1954) because o f i t s s ta lk v lik e base b u t th e io d in e re a c tio n o f th e a s c i is a more im portant d ia g n o stic fe a tu re (D ennis, 1960)#

The co n d itio n s o f th e m obile dunes seem a t f i r s t s i ^ t to be most unfavourable f o r th e n u tr iti o n , growth and

PLATE 2

C. dunensis growing in a s s o c ia tio n w ith A. junceiform e and E. a re n a riu s on the sand- dune in Tentsmuir*

PLATE 3

m

_À

m m m m

■^r-a #

M f r m #

PLATE 4

F s a th y re lla ammophila and E, a re n a riu s on the fo re dune in Tentsmuir*

PLATE 5

-saikN^

humus la y e r underground, in dead wood, in dung o r in ^ m yoorrhisal p a rtn e rsh ip w ith th e liv in g ro o ts o f th e h ig h er p la n ts " . These o b serv atio n s a re very g e n e ra liz e d and th e c u rre n t in v e s tig a tio n was designed to fin d out i f th e fungi were in fa c t more s p e c if ic in t h e i r n u tr itio n a l requirem ents.

The woric com prised th e is o la tio n o f th e fu n g i in c u ltu re , a study of some o f t h e i r growth req u irem en ts, and t h e i r

a b i l i t y to produce e x tr a c e llu la r enzymes which would make them su c c e ssfu l com petitors in th e f ie ld . An attem pt has a lso been made to g et them to form s y n th e tic m ycorrhiza and co n sid e ra tio n has been given to th e d iffe re n c e s

S E C T I O N 2 MATERIALS AND METHODS 2a M a te ria ls

All th e m a te ria ls used in t h is in v e s tig a tio n were c o lle c te d from th e foredunes o f th e N ature Reserve a t Tentsm uir in Fife*

The caryopses o f th e g ra sse s A* a re n a ria . A. junceiform e and E, a re n a riu s were chosen in

November, 1966 when they appeared to be dry. In th e la b o ra to ry th ey were husked and s to re d in clo sed b o ttle s a t room tem perature u n til re q u ire d .

For th e experim ents re q u irin g ro o t e x tra c ts , th e ro o ts were c o lle c te d in August, 196? and th o se req u ired f o r is o la tio n o f fu n g i were c o lle c te d in September,

1966 and May, 1967#

In th e experim ents $a and 5d where sand was used, i t was taken from a depth o f 6-12 inches from about 12

areas a t random where th e g ra sse s and fu n g i flo u ris h e d , and then thoroughly mixed b efo re use*

The f r u i t bodies o f th e fu n g i C. dunensis* M. grammopodia* F s a th y re lla ammophila and P e sisa ammophila were c o lle c te d a t o r n e a r th e beginning of th e i r growing seasons (T able l ) and is o la tio n s made as

Table 1

F ruiting Season

Fungus 1966 ₁₉₆₇

P sath yrella ammophila 1st wk

Aug.-4th wk Nov. 2nd wk Ju ly-3rd wk Nov.

Peziza ammophila 1 st wk

Aug.-4th wk Nov. 2nd wk Ju ly-3rd wk Nov.

M. grammopodia 2nd wk

Aug.-1st wk Dec. 1st wk Dec.3rd wk S ep

t.-C. dunensis 1st wk S ep

t.-1st wk Dec. 1 st wk Dec.3rd wk S ep

t.-Unless otherw ise sta ted the reagents were supplied by B ritish Drug House and were o f Analar grade.

2b General Methods ( i ) S te r ilis a tio n

(a) Glassware

P etri p la te s were s t e r iliz e d with dry heat at 180°0 fo r two hours* Unless otherw ise sta te d , the Brlenmeyer fla sk s were acid washed, rinsed in d is t ille d water,

plugged w ith ncn-absorbent cotton wool and s t e r iliz e d by aut00lavin g at I 3 lb s. pressure per square inch fo r

fifte e n minutes.

(b) Culture media

A ll the culture media used were s t e r iliz e d by autoclaving at I 5 lb s. pressure per square inch fo r f i f ­ teen minutes, except the nitrogen sources. The nitrogen

[image:22.614.91.521.111.325.2]

(o) Caryopses

A fter th e caryopses were husked by hand, th#y were soaked in w ater f o r 1-2 hours and a l l tra c e s of lemma and p a le a were removed w ith a p a ir of fo rcep s an d /o r a s c a lp e l. T his was to allow th e d is in fe c ta n t to reach th e e n tir e su rfa c e o f th e g ra in s . Care was taken not to damage th e embiyos during th e o p e ra tio n .

The caryopses were then soaked overnight in s t e r i ­ liz e d 0 .1 ^ d ifc o ag ar in d i s t i l l e d w ater which acted as a w ettin g agent (C h esters, p erso n al communication 1)66) and then su rfa c e s t e r i l i z e d by soaking in a s a tu ra te d so lu tio n of sodium h y p o c h lo rite which contained 10-14J^ w eight/volum e a v a ila b le c h lo rin e f o r f if te e n minutes# This treatm en t proved too d r a s tic f o r B. a re n a riu s and

A. junceiform e r e s u ltin g in very poor germ ination. The sodium h y p o c h lo rite warn then d ilu te d to $0^ and 10%.

The caryopses were again soaked fo r fifte e n minutes and

then washed in s ix changes of s t e r iliz e d d is t ille d water to remove a ll traces of the s t e r iliz in g agent.

(d) F ru ctifica tio n fo r Isc la tio n

Small p o rtio n s of g i l l s o f young b asid io c a rp s of C. dunensis. M. grammopodia and F s a th y re lla ammophila and se c tio n s of th e hymenium of th e $pothecium of P eziza ammmphii* w srs a s s p tic a lly excised and su rfa c e s t e r i l i z e d w ith yjc sodium h y p o c h lo rite f o r one m inute. The r e s u lts were s im ila r when 1.5% sodium h y p o ch lo rite was used f o r f if te e n minutes#

(e) Roots f o r Is o la tio n

When ro o ts c o lle c te d from T entsm uir were seen by m icroscopic exam ination to be a sso c ia te d w ith

8

f if te e n m inutes and th en washed in s ix changes of s t e r i l i s e d d i s t i l l e d w ater.

( f ) Earthenware P ots and Sand

The new 5" earthenw are flo w er p o ts which were used in th e experim ent to be d escrib ed in S ectio n 5a, were covered w ith aluminium f o i l to prevent them from being flooded during s t e r i l i z a t i o n . They were then s t e r i liz e d by au to clav in g a t 15 lb s . p re ssu re p e r square inch f o r th re e hours.

The s o il used in S e rie s 1 o f th e same experiment was sand which was c o lle c te d from T entsm uir (See th e se c tio n on M a te ria ls). I t was passed through a 2mm mesh sie v e and mixed w ith v e rm ic u lite in th e r a tio of th re e p a r ts sand to one p a rt v e rm ic u lite and then au to - claved a t 15 lb s . p re ssu re f o r fo u r hours. In a d d itio n to au to cla v in g , McArdle (1932) h eated h is sand a t 105*0 f o r s ix hours. I t was found th a t fo u r hours in th e au to clav e was s u f f ic ie n t to k i l l a l l m icro-organism s and so h e a tin g in th e oven was om itted.

The method o f s t e r i l i z a t i o n used f o r S e rie s 2 o f th e same experim ent was d if f e r e n t. m t h i s case, th e sand was not sie v ed and no v e rm ic u lite was added. The p la n t p a r ts found n a tu ra lly in th e sand were in co rp o rated

2b ( i i ) Examination of Roots f o r In fe c tio n Roots from p la n ts in T entsm uir

In September, I966 and May, 19&7 th e underground p a rts o f A# a re n a ria . A* junceiform e and B> a re n a riu s were c o lle c te d from T entsm uir. H oles, about 12 inches deep, were made in th e foredune and th e p la n ts w ith as much o f t h e i r ro o t system as p o s s ib le , were removed and taken to th e la b o ra to ry . The excess sand was removed by washing and th e ro o ts which occupied th e top s ix

inches in th e sand on th e dunes were cut o f f, washed, p laced in d i s t i l l e d w ater and a e ra te d w hile aw aiting

exam ination. These and subsequent ste p s were hastened to prevent changes in th e c e lls e .g . atrophy.

Free-hand lo n g itu d in a l s e c tio n s were made and s ta in e d w ith co tto n b lu e in lacto p h en o l and examined m icro sco p ically in la c to p h e n o l. Between th re e and s ix se c tio n s were made from each ro o t according to th e

l e ngth o f th e p a r tic u la r ro o t. The presence of Basidiom ycetes was recorded when hyphae w ith clamp- connections were observed on o r w ith in th e ro o ts.

Roots o f p la n ts grown in th e greenhouse

These p la n ts were c a re fu lly removed from th e pots and g e n tle shaking removed th e bulk of th e sand. The ro o t system s were then detached from th e a e r ia l p a rts and washed g e n tly to remove as much o f th e sand as p o s s ib le w ithout removing e c to tro p h ic mycelium, i f p re se n t. The prim ary rc o ts were c a re fu lly se p arate d from one an o th er f o r exam ination purposes.

The in d iv id u a l ro o ts were examined under a

10

Tangential lon gitu d in al seotion a were made by

handy stain ed with cotton blue in lactophenol and ex­ amined in lactophenol at X 100 and X 400 m agnifications. An average o f fiv e sectio n s were made from the f ir s t lOom of the roots when a ll that was required was to esta b lish the presence or abswice of the fungus. When in fectio n was observed in roots from one p la n t, the extent of

in fectio n was studied on two o f those roots chosen at random. The roots were very long, reaching a length of ju st over 200cm. They were cut in 10cm lengths and one sectio n made from each length u n til the fungus ceased to be present.

I f the fungus was observed in and/or on the roots, the in fected parts were fix ed in form alin acetic acid -alcohol fix a tiv e and embedded in Parowax follow in g Johanssen*s technique (Johansset^ 1940). Transverse

and lon gitu d in al sectio n s were cut 10-1$

y.

in thickness# Cotton blue in lactophenol was used, as th is sta in

proved superior to magdala red and lig h t green| magdala red and fa st green| picro a n ilin e blue; saffran in and fa s t green. These sectio n s were used fo r more d eta iled study and fo r photography.

2b ( i i i ) Fungal Iso la tio n s from Roots

11

Longitudinal aaotiona of the oortez were then made w ith a eurfaoe s t e r ilis e d blade and 4-5 o f them were put

in each p e tr i p la te and gen tly pushed in to the iso la tio n medium# The sectio n s ranged from l-3mm in length*

Two d iffer en t media were used:»

(a) M artin's medium (Johnson et a l, I 96O)

Agar 20.0 grams

Potassium dihydrogen

phosphate 1*0 "

MgS04#7H20 0 .5 «

Peptone 5*0 •*

Dextrose 10.0 **

D is tille d water 1000 Ml

Rose bengal 35 mg

Streptoqyoin 30 " (D ista Product Ltd. Liverpool)

The rose bengal was d issolved in d is t ille d water and added to the medium before s t e r ilis a t io n . The strepto» myoln was made up in 20% aloohol and added to the medium a fte r s t e r ilis a t io n .

(b) Malt extract agar ♦ dextrose (Raper & Thom, 1949)

Agar 25#0 grams (D ifoo bacto agar)

Malt extract 20.0 ** (Boots)

Dextrose 20.0 **

Peptone 1.0 "

D is tille d

water 1000 ml

The p la te s were incubated at 23^0 and examined daily# When the sectio n s were contaminated by b acteria, t h ^ were washed in streptosQroin (3p mg/lOOO ml) and re-p lated on new media. Sometimes th is had to be repeated two or three tim es before b acteria were com pletely elim inated. On many occasions th is

12

2b ( iv ) Fungal Is o la tio n from F ru c tific a tio n *

C. dunansis was is o la te d in November, 19^5 w hile M. grammopodia. P sath arrella ammophila and P e sisa

ammophila were is o la te d in August, 1966.

The fr u it bodies were removed ca refu lly from the sand-dunes and placed in s t e r ilis e d containers. They were taken to th e laboratory where iso la tio n s were made w ithin tw elve hours. The best r e su lts were obtained when iso la tio n s were made before a tim e lap se o f two hours.

In a ll cases i t was found that is o la tio n s were more su ccessfu l when attempted early in the growing season. I t would appear that as th e growing season progressed the number of Phycomycetes and B acteria which grew from the

inoculants increased and the slow growing Basidiom ycetes were quickly overgrown by the contaminantSë

With the exception of M. grammopodia. the p il e i are vexy th in and g i l l s were used fo r iso la tio n .

Unoontaminated growth was p r a c tic a lly im possible without surface s t e r ilis a t io n and when s t e r iliz a tio n was enough to prevent growth of the contaminants, th e inoculants a lso fa ile d to grow. The same applied to the hymenium o f P esisa ammophila.

The inoculants were s t e r ilis e d as shown previously under " S teriliza tio n " . By means of a s t e r iliz e d loop, thqy were transferred from the s t e r iliz in g agent to p e tr i p la tes containing malt extract agar. This iso ­

la tio n medium had the follow in g constituents#^

Malt extract 37 grams

D ifco agar 20 "

D is tille d water 1000 ml

13

The p la te s were incubated a t 23^0 and were examined p e rio d ic a lly # Growth was observed a f t e r about one week and su b c u ltu re s were made th w , o r as soon as th e hyphae had grown 2»3mm, depending on th e p resence o r absence c f contam inants.

m cases o f P e sis a ammophila. th e spores germ inated in 2-3 days and su b c u ltu re s were made on th e 4 th o r 3^^ day.

2b (v) Growth o f S eedlings

The caryopses were c o lle c te d in November, 1966 and germ inated between January and A p ril, I967#

S eedlings were o btained under two d if fe re n t condi­ tio n s . Those which were used in th e in o c u la tio n experi­ ment to be d escrib ed in S ectio n 3a, were grown on f i l t e r paper in c r y s ta llis in g d ish e s, w hile th o se used in

Experiment 3& - p roduction o f ro o t exudates and t h e i r e ffe c t on th e growth o f is o la te s - were grown in

s t e r i l i s e d sand in one l i t r e b eak ers.

Four 9om Whatman No. 4 f i l t e r papers were to m in sm all p ie c e s and p laced in each c r y s ta lliz in g d ish . T h irty ml d i s t i l l e d w ater were added and two whole 9om

f i l t e r paper d is c s were put on to p o f th e to m ones. T his was to in c re a se th e amount of w ater in th e dishes w ithout th e r is k o f drowning th e "seeds*. The d ish es were then covered w ith a l i d and s t e r i l i z e d as

d escrib ed under S ection 2 b ( i)( b ) .

14

daric except when lig h t was adm itted during exam ination.

As soon as they had germinated they were transferred to a window s i l l at room teaq>erature.

For th e ro o t exudate experim ent, th e caryopses were su rfa c e s t e r i l i s e d as d escrib ed in S ectio n 2 b ( i)( c ) and were put to germ inate in one l i t r e beakers -f f u l l of s t e r i l i z e d sand# The beakers were then put under la rg e b e ll ja r s in a warm dark room u n t i l germ ination commenced a f t e r which tim e they were given a r t i f i c i a l lig h t f o r n in e hours d a ily .

The s id e of th e b e ll j a r and th e sh eet o f g la s s on which i t re s te d were swabbed w ith 70% alco h o l. The b e ll

ja r s were used to prev en t ex cessiv e ev ap o ratio n . I t was hoped th a t t h i s would a lso keep out a i r borne fungal and b a c te r ia l spores and so keep th e se ed lin g s as

15

S E C T I O H ï

MORPHOLOaiCAL MTO CULTOHAL CHAHACTERETICS Qg THE E O U T ffi

A.

Conocarb. dunm ali

This i s a r e la tiv e ly slow growing fungus# At 2}*C th e average growth r a te on 2% m alt e x tra c t agetr is 3*7mm p e r week# The mycelium appears «diite w ith vexy l i t t l e a e r i a l hyphae# On a g a r p la te s th e fUngal colony i s o fte n zoned# Clamp connections a re p resen t# Sometimes s tr a ig h t and

narrow hyphae may he p re se n t b a t o s o a lly th ey become balboas ju s t behind th e growing p o in t - a b o lb itia o e o a s c h a ra c te r­ i s t i c ( P la te s 6 and 6a)#

WelaiiQlaao^

This i s a ls o a slow

growing

fongas

incroEksing a t

th e

ra te

o f 3»5mm p e r week

on

2%

malt ex tract ag^r a t

23 C# A e ria l hyphae a re absent# I t a ls o has la rg e hyphal pro­

tu b eran ces (P la te s 7 and 7a) and clamp connections a re absent# I t causes th e a g a r in i t s v ic in ity to become

b la c k is h , perhaps because o f e x c re tio n o f su b stan ces in th e agar#

c.

.amOThila

This grows r e la tiv e ly f a s t and under c u ltu r a l condi­ tio n s s im ila r to th o se o f C^uSSBSBSlai I t grows a t an average o f X0#4mm p e r week# A e ria l hyphae a r e sh o rt and dense; a t f i r s t th e hyphae a re w hite b u t th ey become lig h t brown w ith age# A ll hyphae a re branched and s t r a i ^ t and

PLATE 6

A colony o f C. dunensis growing on m alt e x tra c t a g a r.

PLATS 8a

y.L i-l

m

r

&

oo

PLATS 7

A colony o f M. grammopodia growing on malt extract

agar# The c h a r a c te r istic d isco lo ra tio n o f the agar i s c le a r ly demonstrated.

PLATS 7a ( i )

PLATT) 7a( i l)

FLATS 8

P s a th y re lla ammophila growing on m alt e x tra c t ag ar f o r two weeks*

«LATE 8a

c.

This i s a f h s t growing BisoonQroete and in c re a se s i t s ra d iu s a t an average r a te o f 2Qom p e r week under th e condi­ tio n s d escrib ed fo r th e BasidiOBQroetee# A e ria l hyphae a re sh o rty w h itish and th in . A m gal co lo n ies a re alw ays soned on a ^ r p la te s .

S u b cu ltu res had to be made every two weeks t otherw ise th e fungus f a ile d to grow. Sometimes up to 20 su b c u ltu re s were made from th e same colony and only one o r two grew# Sometimes a l l I k ile d to grow and th e is o la tio n was re a d ily

lo s t# This was th e case when su b c u ltu re s were made from co lo n ies two o r more weeks o ld . E ith e r th e fungus produces s ta lin g pro d u cts which prev en t th e hyphae from growing on fre s h ag^ry o r th e medium i s q u ick ly d e p le te d o f some essen­ t i a l growth substance which i t cannot sy n th e sise f o r i ts e lf # E . E J

This i s a Bas idiom ycet e which was is o la te d from ro o ts o f EItous aren arlt^s in S ept ember y 1)66 u sin g th e technique

d escrib ed in S e c tio n 2 b (iii} «

The fungus was con fin ed to th e c o r tic a l c e lls o f th e o ld e r p a rts o f th e ro o ts both i n t e r - and i n t r a - o e llu la r ly . I t was never lo c a te d in ro o t h a irs n o r on th e o u tsid e o f th e r o o ts . In th e c o rte x th e hyphae were s t r a i ^ t and branched and alm ost a l l se p ta had v is i b le clamp connections*

17

growth# This new development was w h ite and o<»paot#

On exam ining th e colony m icrosoopioallyy i t was found th a t th e hyphae in th e primaxy growth were c h ie f ly narrow and e t r a i ^ t idiiXe th o se in th e eeoondaxy growth were bulbous and much b ro ad er (P la te s 9 and 10)#

When ro o ts o f a r e m r ia aad At j B S a i . t a n i were in o c u la te d w ith t h i s fUngusy i t s morphology was ag a in d if f e r e n t ( P la te ll) # I t was much more bulbous th an when i t was grown in c u ltu re #

P# A 1 and A 2

These a r e two fias idiom ycet es which were is o la te d from Ammophila ro o ts in May y 1967# u sin g th e tech n iq u e d escrib ed in S e c tio n 2 b ( iii) #

As in th e case o f E 1 th e hyphae were confined to th e o o r tio a l c e lls both in te x ^ and i n t r a - o e llu la r ly and were n ev er found in young ro o ts $ in young p a r ts o f o ld e r ro o ts n o r

in th e sm all l a t e r a l ro o ts# A 1 and A 2 appeared to grow to g e th e r b u t A 2 extended f u rth e r down th e le n g th o f th e ro o t th a n A 1 idiioh was confined to th e u p p er th ir d o f th e roots#

In P la te 12 y A 1 i s seen a s bulbous hyphae in th e c e lls o f th e c o rte x w hile A 2 is ju s t v is i b le a s th in lin w # P la te s 13 and 14 show more d e ta ils o f t h e i r s tru c tu re #

On m alt e x tra c t a g a r a t 23*Gy A 1 was a r e la tiv e ly slow grower - average in c re a se o f 2#6mm p e r week w h ile A 2 grew a t 4#6mm in th e same tim e*

In A 1 th e hyphae appeared brownishy no a e r i a l hyphae were v is ib le and m ic ro sco p ic ally th e hyphae were bulbous. S epta and olamp connections were re a d ily seen#

PLATS 9

Camera lu c id a drawing o f hyphal t i p s o f E 1 taken from the growing edge o f a th re e week o ld colony*.

FLATS 10

PLATE 11

Non - bulbous hyphae o f E 1 in th e c o r tic a l c e lls o f E. a re n a riu s which was c o lle c te d from Tentsmuir* Compare th is w ith P la te 59 where th e hyphae are bulbous * ( X IQOO ) •

PLATE 12

y

flî _{TH£ C.BI.U-}OUTU|A/£ OP

y

f

.1

I

PLATE 13

A 1 in c o r tic a l c e lls o f A# a re n a ria (xlOOO)

PLATE 13a

c

#

PLATE 14

...

-.1.

1.Ô

P h y slo lo g lo a lly th ey were a lik e in t h e i r v itam in and

n itro g e n reqnirem ente, th e q u a n titie s o f o e llu lo ly tic enaymee produced in v i t r o and th e amount o f p o ly # ien o l oxidase which th e y produced on ta n n ic and g a llic a c id m edia. However#

th e y d if fe re d in th e p ro d u ctio n o f p e o tin o ly tio enaymes and in th e stim u la tio n o f t h e i r growth hy e x tra c ts from th e ro o ts o f

A . A . A m o ^ ifo n n e ThflOT » r e

probably two s tr a in s o f one and th e same fungus and t h e ir

d iffe re n c e could be due to th e A c t th a t B 1 had been is o la te d e i ^ t months b efo re A 1 and might have undergone changes in i t s p hysiology.

Cn 2^ m alt e x tra c t a g a r dunm m is# lU grammopodia. B 1 and A 1 in v a ria b ly produced bulbous hyphae# A 1 a ls o appeared bulbous in s i t u in th e ro o ts o f A. a r e n a r ia . The hyphae o f B 1 were not o r ig in a lly bulbous in th e ro o ts o f B. a re n a riu s a t th e tim e o f is o la tio n b u t idien on 2 $ m alt

e x tra c t a g a r and when th e is o la te re in fe c te d th e ro o ts o f th e th re e g ra s s e s # th ey were bulbous. T his i s perhaps a

phenomenon which re q u ire s f u rth e r m ention.

Wat lin g (p erso n al communication# I967) has found th a t th e bulbousness o f b o lb itia c e o u s sp e c ie s in c re ase d w ith th e amount o f su cro se in o o rp o rated in th e medium. He suggests th a t i t i s r e la te d to osm otic p re s s u re . As no experim ental d a ta i s a v a ila b le i t cannot be s ta te d th a t h is ex p lan atio n a p p lie s to th e se fungi u n der in v e s tig a tio n .

Swollen o r bulbous hyphae have been re p o rte d a t le a s t in two o th e r groups o f fu n g i i . e . Oonrinus and P é n ic illiu m .

M adelin (196O) o ffe re d an e:rplanation fo r th e occurrence o f sw ollen c e lls in th e v e g e ta tiv e mycelium o f Conrinue

19

a v ery la rg e p ro p o rtio n o f th e c e lle w ith in th e a g a r a t

d ista n c e s g r e a te r th a n 5«m from th e m argin were v a rio u s ly in f la te d and g ro ssly d is to r te d and were com pletely f i l l e d w ith dense and h ig h ly r e f r a c tiv e c o n te n ts . D esp ite th e sw elling# olamp connections were g e n e ra lly c le a r ly v is i b le . (T his a ls o h o ld s tru e f o r Conocvbe d u n en sis. f o r A 1 and f o r E l ) . These in f la te d c e lls were a b se n t in a e r i a l hyphae.

Between th e 11th and th e l6 th days# when sporophores were growing and m aturing# M adelin observed v is i b le changes w ith in th e sw ollen c e lls — vacuoles appeared and grew pro­

g re s s iv e ly la r g e r w hile th e amount o f deeply s ta in in g cytoplasm p ro p o rtio n a lly dim inished. The sp en t c e lls re ta in e d t h e i r shapes b u t lo s t t h e i r s ta in in g p ro p e rty . These changes were a ls o observed on h a p lo id mycelium which u s u a lly f r u ite d

ta r d il y and abnorm ally# i f a t a l l . As a r e s u lt o f th e se o b serv atio n s y M adelin suggested th a t th e sw ollen c e lls played an im portant p a r t in re se rv in g food f o r f r u itin g .

Conooarbe dunensis produced f r u i t body prim ordia on m alt e x tra c t a g i r a f t e r about th re e weeks b u t only sm all abnormal f r u i t bodies have been produced on t h i s medium. Swollen hyphae were observed alm ost a s soon a s th ey s ta r te d growing. So A r# th e o th e r fungi w ith bulbous hyphae have not been

observed to f r u i t in c u ltu r e . Perhaps th ey can go as A r a s s to r in g up food re se rv e s but th e medium lacks some A c to r which th ey need fo r f r u i t i n g .

Bent and Morton (1963) observed sw ollen hyphae in sub­ merged c u ltu re o f P é n ic illiu m g riseo A lv n m . T h eir form ation was regarded by Lilly and B arn ett ( l9 5 l) a s response to one o f se v e ra l co n d itio n s u n A v o u rab le to growth e .g . low pH#

20

presence o f in h ib ito r s . Bent and Morton (1963) found th a t th e ** la rg e c e lls were m orpholcgioally and developm entally eq u iv ale n t to sporophores in which th e noxmal development le a d in g to c o n id ia l form ation is n o t com pleted owing to some m etabolic derangement caused by an abnoxm ally low pH o f th e medium" .

A lth o u ^ th e r e la tio n s h ip between la rg e c e lls and

21

S E C T I O H A

PHYSIOLOGICAL CHAmCTERETICS OP MB ISOLATES

4a V itam in requirem ent

This experim ent was condaoted w ith a view to

a s c e r ta in in g w hether th e fhngi were a b le to sy n th e siz e t h e i r own v itam in s o r %#hether th e s e had to be su p p lied from an e x te rn a l so u rc e . The fo llo w in g fu n g i were su b je c te d to th e t e s t s - C. d u n en sis. lU ^rammopodla.

P s a th y re lla ammophite. ^

A 2. They were grown f o r 2-3 weeks on a medium oon^ ta in in g 2ÿ d ifc o a g a r and IgG glucose to e lim in a te th e p o s s i b ilit y o f them c a rry in g o v er tra c e s o f vitam ins w ith them .

MsEtàaà

The v itam in s thiam ine (lOO p g /li tr e ) # b io tin (5 yL^f l i t r e ) and p y rid cx in e (lOO ju g ^ litre ) f o r which filam en­ to u s fUngi a re most fre q u e n tly d e fic ie n t# were su p p lied to seven s e r ie s o f c u ltu re media in th e fo llo w in g

manners—

S o lu tio n 1 m b a s a l medium a c tin g a s a c o n tro l " 2 - " " + thiam ine (B .S.HiLGK)

"

3 - "

" + h iotin (B.D.HILG»)

" 4 " " " + p y rid o zin e (B.B.HtLGR)

3 ■ ^ thiam ine and b io tin ** 6 » ^ ** *f thiam ine and p y rid o x in s ” 7 • ” " + b io tin and p y rid cx in e

" 8 « ** " + thiam ine# b io tin # p y rid cx in e The b a sa l s y n th e tic c u ltu re medium was th e glucose — a sp a ra g in e medium o f L illy and B arn e tt ( l9 3 l) uud had

-22

Glucose

A sparagine

KH2PO4

II^04.7H 20

Pe"^ Zn^ Ma^

D is ti lle d w ater

(B.D.H:LQR) (B.B.HtLCER) 20 .0 grams

2 .0 «

1.0 ••

0 .5 " 0.2 mg 0.2 mg 0.1 mg 1000 ml

pH a d ju ste d to 6 b efo re s t e r i l i s a t i o n

The b a s a l medium was s t e r i l i z e d and when cooled th e v itam in s were added a t th e r a te in d ic a te d above. The

thiam ine and p y rid o zin e were made up in 70^ alco h o l (sto c k s o lu tio n s co n tain ed lOkng^ 100ml) and s to re d in a r e f r ig e r a to r u n t i l needed. When 20^ a lc o h o l ( L illy and B arn ett# 1951) was used# contam ination was observed a f t e r th e fla s k s w ith th e in o c u la n ts had been incubated

fo r e ig h t days. There was no such problem when 7(% alc o h o l i#as used in s te a d o f 20^. The b io tin was b o u ^ t

in 1ml ampoules c o n ta in in g 1 pg* The media were

dispensed in 25ml q u a n titie s in th e p re v io u sly s t e r i l i z e d lOGmi Erlenm eyer fla s k s and were in o c u la te d and incubated a t 23*0. The experim ent was s e t up in d u p lic a te .

In th is # a s in a l l q u a n tita tiv e experim ents in

th is in v e stig a tio n # equal s iz e in o c u la n ts were u sed . A 5mm sq u are was o u tlin e d on a sh e e t o f graph p ap er. Each p e t r i p la te from which th e in o c u la n ts were taken# was superim posed on th e graph paper so th a t th e growing edge o f th e colony co in cid ed w ith one s id e o f th e square#

The in o c u la n ts were th e n out cu t 5™ square and approxi­

23

A fte r te n weeks th e m yoelia were rin s e d in d i s t i l l e d water# tr a n s f e r r e d to p re v io u sly weighed aluminium f o i l b o ats and d rie d a t 50% to co n sta n t w e i^ t# T h eir dry

w eights were th en determ ined#

The r e s u lts shown in Table 2 have been c a lc u la te d a s p ercen tag es o f th e c o n tro l and a r e expressed in P la te s 15-21# From Table 2 and P la te 15» i t can be seen th a t

O# dunensis i s a b le to grow on th e b a s a l medium b u t th e a d d itio n o f thiam ine and pyridoxins# s in g ly o r to g eth er#

caused a marked in c re a se in growth# B io tin alo n e caused a d ecrease in growth# b u t th e a d d itio n o f

thiam ine was e f f e c tiv e and doubled th e grow th. When b io tin and p y rid o x in e were in th e same medium# growth was e q u iv a le n t to th a t cn th e b a s a l medium. When a l l th re e v itam in s were to g eth er# growth was a s good a s w ith thiam ine on i t s own but s li g h t l y le s s th an w ith p yrid o x in e alone# th iam in e e p y rid o x in e o r w ith thiam ine and b io tin to g eth er#

It^ th erefo re^ appears th a t thiam ine and pyridoxine

must be added to th e s u b s tra te f o r th e h e a lth y growth o f b u t th a t b io tin on i t s own o r w ith pyrid o x in e has an in h ib ito ry e f f e c t .

I

g

I

_s

A

r4

iH iH

CM

•H

r4

•H

00 NO

25

B io tin , Iqr i t s e l f , p e rm itte d growth which was n e a rly tw ice th a t on th e b a s a l medium» Growth was reduced to n e a rly h a l f th a t on th e b a s a l medium when pyridoxine alo n e was added#

T h erefo re , M» grammopodia grows b e t t e r w ith an e x te rn a l supply o f thiam ine o r b io tin b u t i t would ap p ear th a t p y rid o x in e in te rf e r e d w ith th e stim u la to ry

e f f e c t o f b io tin when they were b o th in th e same medium# P s a th y re lla ammochlla grew v ery p o o rly on th e

b a sa l medium but th e a d d itio n o f thiam ine g re a tly in c re a se d th e growth to more th a n sevenfold# B io tin

by

i t s e l f was e f f e c tiv e in cau sin g n e a rly th re e fo ld in c re a se whereas p y rid o x in e p e rm itte d only tw ofold increase# Thiamine and b io tin to g e th e r were a s

e f f e c tiv e a s p y rid o x in e alo n e b u t when a l l th re e were to g e th e r growth was stim u la te d to th re e tim es th a t on th e b a s a l medium# Thiamine + p y rid o x in e p erm itted growth which was ju s t b e t te r th a n th a t on th e b a sa l medium b u t s lig h tly p o o rer th an p y rid o x in e + b io tin #

Growth o f P s a th y re lla aam onhila i s , th e re fo re , stim u la te d

by

an e x te rn a l supply o f each o f th e th re e v itam in s p a r tic u la r ly by thiam ine#

Pgnlmm MWPBhite

26

This ftingas a ls o re q u ire s an e x te rn a l supply o f thiam ine and b io tin in o rd er f o r i t to produce good grow th. I t is probably a b le to sy n th e sise i t s own p y rid o x in e.

The b a s a l medium supported vezy good growth w ith

t h is fungus a l t h o u ^ th e a d d itio n o f thiam ine o r

p y rid o x in e produced an in c re a s e . When b io tin was th e only v ita m in p re se n t growth was d ep ressed . The a d d itio n o f thiam ine p lu s b io tin gave b e t t e r growth th a n th a t

o b tain ed when th ey were added s in g ly . Thiamine plus p y rid o x in e had a combined e f f e c t o f in c re a sin g th e growth to more th an tw ice th a t o b tain ed on th e b a s a l medium.

The com bination o f b io tin w ith p y rid o x in e had a reducing e f f e c t b u t when thiam ine a ls o was added growth in c re a se d .

B iotin# th e re fo re # seems to have an in h ib ito x y e f f e c t w h ile thiam ine and p y rid o x in e stim u la te d growth,

à j i

In A 2 . thiam ine#w hether s in g ly o r combined#

markedly in c re a se d th e growth# B io tin caused a d ecrease W iile p y rid o x in e had no e ffe c t# Growth in c re a se d when p ÿ rid o x in e and b io tin were added together#

The growth o f t h i s fungus is# th e re fo re # stim u la te d by an e x te rn a l supply o f th ia m in e ,

£JL

E 1 grew on th e b a s a l medium b u t th e a d d itio n o f thiam ine p erm itted much b e t te r growth# B io tin was in h ib ito ry but th e a d d itio n o f thiam ine to b io tin gave b e t te r growth th a n was ob tain ed w ith thiam ine alone#

z < c c_D TJ a _o o c o U iS t§

i§ 8

r

-s

ÿ

_c

V u (Ü c O U Cl c Ê_w j= H O CD V c X o TO Q_ 08

V C

.E % E o .5 IE

_c

"5 g ë g

I I

_'P

Q-«0g

II

. * 1

l i

I -z a: 5

a

z < Q. O E E n N N QJ Q-§ a 8 8 § § C O U V c Ê w s: I -o CO V c X o T7 Q_ 00

V C

.£ w_{E o}

£ 2

II

H

o8«

a

_{H- -o}oB«

Z -D LU O

I I

O (X) .8

.8 y .8 is c o

u cûo

V c X o TJ a .

06 g

^ 1 E "c - x> 2

_c

00

(U c c -

Ê o

°0c

Sx 0-2 û. c û ^

06 ^

a - c c X

z z oc:

O 3:

Q_

o

E E

Z r

< 5

— û_ _O

/ L.

a

z

LU

Z

LU

0^ 5

a

LU

z

LU

Z

LU

D

z

LU

Z oc

D

O_LU

aC _

LU

Z

< 18_{% u}c o

c o

u Cûo

V

c

X

o

•D_C

û_

00 00 « og _{C ^ .5}00 g

.E Z

I S

E =%

£.2 Ê ? 53 2 ^ .2 2^ %

27

tw ice th a t on th e h asal mediun* AgEtin pyridoxine had l i t t l e e ffe c t tdien combined w ith b io tin bat a l l three together perm itted eome growth*

T his IbngoBf th e re fo r e , grows nuoh b e t t e r when th e re i s an a d d itio n o f thiam ine#

D isooasioa

Fungi re q u ire v itam in s f o r grow th, rep ro d u ctio n and o th e r v i t a l functions# C e rta in ones must o b ta in one o r more o f th e v itam in s from th e s u b s tr a te , e#g# Phvoomyoes

re q u ire s th ia m in e | o th e rs a r e a b le to sy n th e siz e from th e compounds o f th e medium a l l th e v itam in s in s u f f ic ie n t q u a n titie s to meet t h e i r needs

e.g# A sp e rg illu s n ic e r # From th e experim ent d escrib ed above, I t can be seen th a t a l l seven fbngi responded

by in c re a se d growth to a d d itio n o f th iam in e , fo u r o f them to a d d itio n o f b io ti n and only two to p y rid o x in e.

T his experim ent, however, was only a "sc re e n in g te s t" and th e r e s u lts need to be v e r if ie d u n d er more c o n tro lle d co n ditions# For example, th e ohmnioals

should be p u r ifie d p r io r to u se to av oid th e r is k o f con­ tam in atio n by s ig i if i o a n t amounts o f th e vitam ins# The experim ent should a ls o run over a co n sid erab le p erio d o f tim e because fu n g i change d u rin g development in t h e i r s y n th e tic oap ao ity and in t h e i r v itam in needs (Cochrane, 1958).

A nother problem i s th a t d eterm in atio n should be made

over a range o f c o n c e n tra tio n o f th e v ita m in s. I t has

been assumed h ere th a t optimum o r n e a r optimum amounts

28

n ea r optimum fo r many filam entous fUngi ( L illy and

B a rn e tt, 1951)# However, th e optimum amount o f a v itam in may v ary w ith dhanges in o th e r c o n d itio n s and may a ls o he d if f e r e n t fo r d if f e r e n t fungi#

The experim ent a ls o su g g ests th a t b io tin had a d e p re ssin g e f f e c t on growth o f C# d u n en sis# E 1. A 1

and A 2 and p y rid o x in e on th e growth o f N. cmmmooodia#

This may be due to an ex cessiv e dosage o f th e v itam in (M athur, B arn ett and L illy , 1950; L illy and B a rn e tt,

1951) o r th e r e s u lt may be in flu en c ed by some environ* m ental condition# I t was found (B arn ett and L illy ,

1948) th a t in S o le ro tin ia o am elliae th e same concentra­ tio n o f in o s ito l which stim u la te d growth a t 26*0 caused an in h ib ito ry e f f e c t a t 2?*C# B io tin and pyridoxine were a ls o found to reduce th e growth o f D iplooarpon

(Shirakaw a, 1955)# Shirakaw a found th a t th e a d d itio n o f b io tin and p y rid o x in e , sin g ly o r in com­ b in a tio n ,to a medium c o n ta in in g thiam ine depressed

growth u n le ss in o s ito l was p resen t# B# ro sea i s

t o t a l l y d e f ic ie n t f o r thiam ine and p a r t i a l l y d e fic ie n t f o r in o s ito l# P rie s (1943) te s te d th e e f f e c t o f a v ery s tro n g p y rid o x in e c o n c e n tra tio n on th e growth o f v ario u s Asoomyoetes, but no to x ic e ffe c t could w ith

c e r ta in ty be proved in any case#

29

For maximam growth to ta k e plaoe# th e re moat be a baXaaoe between n a t r i t l o n a l and environm ental Ih o to rs and th e d if f e r e n t v itam in e , sin c e growth I s a r e s u lt o f

In te x a c tin g fh c to rs among idiloh a r e th e vitam ins#

V eg etativ e growth m easured by dxy w e i ^ t Is ap p a re n tly th e

most u s e fu l o r lte r lo n o f th e u t i l i s a t i o n o f vitam ins alth o u g h rep ro d u ctio n and o th e r p ro cesses a re a lso

a ffe o te d and th e fu n g i m l ^ t e x h ib it t h e i r d e flo le n o le s fo r p a r tic u la r v ita m in s d u rin g th e s e p ro cesses r a th e r th an in t h e i r v e g e ta tiv e stage#

The v itam in s re q u ire d by th e se fUngl a s demon­ s tr a te d by th is in v e s tig a tio n a r e probably su p p lied by th e liv in g ro o ts In a s s o c ia tio n w ith which th ey grow o r from th e decomposing p la n t and anim al m a te ria ls p rese n t in th e sand# The fu n g i m l ^ t a ls o be a b le to

sy n th e s is e t h e i r own supply o f v itam in s when in t h e i r n a tu ra l environm ent alth o u g h u n ab le to do so under th e c o n d itio n s o f th e p re se n t experim ent# I t cannot be overem phasised th a t th e p a tte r n o f growth e x h ib ite d by th e se fu n g i m l ^ t be com pletely d if f e r e n t when th e experim ental c o n d itio n s a re v aried # The seem ing v itam in d e fic ie n c ie s must be confirm ed u s in g d if f e r e n t

30

4b U tilization

at Nitrogen S<mroea

T his experim ent was c a rr ie d o a t to detexm ine which oompound(s) could be u t i l i z e d a s B ouroe(s) o f n itro g e n by G . d u n en sis. graaaopodia# P s a th y re lla anm ophlla.

£ Jk f k A t PQlTOoros b e tu lin u s and

Panaeolus sem l-o v atu s. th e l a s t two b ein g used as t e s t fh n g i. These were chosen beoause th ey have q u ite

d if f e r e n t n u tr itio n a l requirem ents from th e o th e r fhngl and th e y were r e a d ily a v a ila b le # P# b e tu lln u s was k in d ly su p p lied by th e Botany Department c u ltu re s to r e and P. semiwovatus (w ild ty p e) was su p p lied by Mrs# K rinos in th e Honours C lass in th e same departm ent# Method

The fh s t growing members, th a t I s , P ezlza ammophlla#

mmWPhAM# Polyporos an a Panaeolaa

aem l-ovatus were grown f o r one week on m alt e x tra c t a g a r a t 23*C W iile th e o th e rs were c u ltu re d f o r up to th re e weeks b e fo re th ey were used a s Inoculants#

Seven n itro g e n sources (T able 3) were s e p a ra te ly In ­ co rp o rated In th e b a s a l medium suggested by L illy and B arn ett (1951) which contained no chem ically combined n itro g e n o th e r th a n any im p u ritie s which m l ^ t be p re se n t in th e c o n s titu e n ts used# The w eights o f th e compounds used were c a lc u la te d to c o n ta in a w ei# it o f n itro g e n

é q u iv a la it to th a t in 2 grams o f a sp arag in e ( L illy add B a rn e tt I l)5 l)#

The ex>^rlm ent was s e t up in d u p lic a te and th e media were d ispensed in 25 ml q u a n titie s In p re v io u sly s t e r i ­

liz e d 100 ml Erlenm eyer flask s# The medium was a d ju ste d

to pH 6 b e fo re s te r iliz a tio n #

31

N itrogen sources H e i^ t in g ra m e /litre

Potassium n i t r a t e KHO^ 2.69

Ammonium t a r t r a t e 2.45

d l A lanine CHj .CH(HH2)COOH _2.37

d l A sparagine NHgCO.cHgCaCimg ) .cooh 2.00

G lycine MHg.CHg.COOH 2.00

d l M ethionine CEgfSCa iCHg.CHCNHgi.OOOR _3.97

Urea MHg.CO.HHg _{0 . 2 0}

32

th ey could get fre e oxrgen# Ihoubation a t 23 C la s te d

fo r e i # i t weeks a f t e r which tim e th e r e s u lta n t m y celial mats were washed, d rie d a t gO^C to co n stan t w e i ^ t and

w e ir e d . T h eir mean dry w eights a re shown in Table 4*

R esu lts

The r e s u lts f o r a l l th e fu n g i were tak en from a s in g le experim ent which should r u le out b o th v a r ia tio n in th e b a s a l medium and th e age and v ig o u r o f th e

inoculum# % e p ercen tag es o f th e c o n tro l were calcu­ la te d and th e r e s u lts a re shown in th e form o f

histogram s#

From Table 4 and P la te s 22-29, i t can be seen th a t M. /rrammopodla and P eziaa ammophlla u t i l i z e d a sp arag in e b e s t o f a l l th e seven sources o f n itro g e n

su p p lied to them# In a d d itio n Conocvbe a ls o had good growth on ammonium n itro g e n and th e amino a c id s a la n in e and glycine# Growth on u rea was b e t te r th an th a t on

th e c o n tro l whereas on n i t r a t e and m ethionine growth was poorer#

The r e s u lts f o r M. grammooodia were alm ost s im ila r to th o se o f c . but h e re growth on u re a was alm ost a s good a s th a t on g ly c in e o r on ammonium n itr o ­

gen but b e t te r th an on alan in e#

^mmoohlla# however, was q u ite d iffe re n t# % e n i t r a t e n itro g e n was second only to asparagine#

A lanine a ls o supported some grow th, g ly c in e le s s ; ammonium n itro g e n and m ethionine were below control#

In P s a th v re lla ammoohila. ammonium t a r t r a t e and u re a were th e only two sources to support growth# A ll