Substrate decomposition and product release by ericoid and ectomycorrhizal fungi grown on protein

Agriculture, E c o s y s t e m s and Envlronment, 2 8 (1989) 285-291 Elsevler Science Publishers B.V., Amsterdam Printed in Czechoslovakia

SUBSTRATE DECOMPOSITION AND PRODUCT RELEASE BY

ERICOID AND ECTOMYCORRNIZAL FUNGI GROWN ON PROTEIN

By A.R. LANGDALg and D.J. READ Department of Plant Sciences,

ABST~CT liquid

The e r i c o i d

of

harvests

as the sole

o f one s e t o f c u l t u r e s

exosenous

Sheffield

and an e c t o m y c o r r h i z a l

medium w i t h p r o t e i n

exception

The U n i v e r s i t y ,

glucose.

Dry

f u n g u s were e a c h grown i n

source

of the ericoid

weight

yields

and t h e p r o d u c t s o f p r o t e i n

of nitrogen endophyte,

were

medimm. No amaonimm was d e t e c t e d

the

of

analyses

Results

o f amino a c i d r e l e a s e

protein

utilisation

~NTROD~CTION

It i s known t h a t e r i c o i d

can be t r a n s f e r r e d

Abuzlnadah

tional

significance

protein

breakdown.

(HysenoscYvhue e r i c a e )

sequential

of amino-acids

and

When E. e r i c a e

was grown i n

and g r o w t h c e a s e d and e c o l o g i c a l

discussed.

and t h a t

to infected

f u n g i can

the nitrogen

host plants.

released (Eajwa e_t_t

& Read 1986 a ) .

Because of their

potential

it

to

the

is

The

necessary

pattern

~nd an e c t n -

of

characterise

protein

before

quantitative

and some e c t o m y c o r r h i z a l

as sole source of nitrogen

from t h e a u b s t r a t e a l . 1985,

are briefly

the

Growth o f b o t h

was c o m p l e t e . The p h y s i o l o g i c a l

of these observations

protein

at

in these cultures

ammonium was r e l e a s e d

complications

utilise

measured

qualitative

are presented.

the absence of exogenous glucose before

of

with

in the presence

to the release

into the culture

growth.

and,

breakdown d e t e r m i n e d .

fungi in the presence of glucose led rapidly

termination

S10 2TN, UK

utilisation

by

(Hebeloma c r u s t u l i n i f o r m e )

nutri-

products an

of

ericold

mycorrhizal

2 86 fungus has now been examined protein

breakdown

and the identities

have been determined

of the major products of

in H. crustullniforme

using HPLC

analysis of sequentially harvested culture filtrates.

MATERIALS

AND METHODS

temperature

Both

on a basal

fungi were grown in liquid culture

medium

(full

strength

MMN,

minus

at room

yeast

extract,

plus lOg glucose litre -1) in which the pure protein bovine serum albumin (BSA)

- M.W.

Three

replicate

tlme

determined

protein.

67,000.

N content

cultures by

the

the sole source of nitrogen.

of each

fungus were harvested

pattern

of

fungal

growth

at set points in

and

utilisation

of

The culture solutions were aseptically filtered by millipore and

the funsal yield was determined c o n c e n t r a t i o n of t h e f i l t r a t e Chemical

16% - was

Company) and

the

derivatisation

w i t h 0PA.

after

protein

residual

by oven drying at 80"C.

Total

protein

was measured u s i n g Coomassie r e a g e n t

(Pierce

amino-acid

content

determined

by HPLC a f t e r

Ammonium was d e t e r m i n e d u s i n g M e s s l e r ' s

had been removed w i t h 10Z TCA.

reagent

The above e x p e r i -

ment was r e p e a t e d w i t h ~. e r i c a e but u s i n g m o d i f i e d HNN w i t h o u t g l u c o s e .

RESULTS

A. H v l e n o m c ~ h u s e r i c a e

The a b i l i t y

of the e r i c o i d

s o u r c e was c o n f i r m e d . over the f i r s t

rate days.

declined

in

of

the

inoculation,

protein

quantities

solution

by

but

protein

this

4,

but

remained, their

rapidly

growth b e i n g a s s o c i a t e d (Fig. i ) .

Growth

breakdown c o n t i n u e d u n t i l ,

of s u b s t r a t a day

as a s o l e N

fungus increased

of a p p r o x i m a t e l y 50Z of t h e p r o t e i n

from 8 days

negligible

detectable

Dry w e i g h t y i e l d

e i g h t days a f t e r

with the u t i l i s a t i o n

e n d o p h y t e to u t i l i s e

by 24

Free amino a c i d s were

concentration

increased

287

markedly as growth slowed from day 12 up to day 24 at which point there was,

for the first time, a decline.

This decline may be attributable to

the fact that protein levels at this stage were no longer sufficient to support growth.

Free ammonium was not detected at any stage during the

development of the cultures.

In medium,

NH4*

the it

absence being

reached

of

first

free

glucose,

detected

after

18 ~g m1-1 by 12 d a y s .

ammonium was 4 days of

At t h i s

incubation.

stage

glucose

to

these

ammonium and a f u r t h e r

B. Mebeloma

(i)

fall

led

to

complete

protein

crustuliniforme f u n g u s were

fall

in p r o t e i n was

utilisation

and

protein

b o t h slow o v e r t h e

A s u r g e of growth

fungus

of

the

free

amino

utilisation

first

40

utillsed.

days.

they

were

in

release

the

by

H.

ectomycorrhisal

24 days f o l l o w i n g i n o c u l a t i o n

(Fig.

d u r i n g which a p p r o x i m a t e l y 70Z of t h e added

This

Thereafter that

being

growth of t h e f u n g u s .

acid

from 24 to 48 days was a s s o c i a t e d w i t h a d r a m a t i c

concentration

s h a r p l y which s u g g e s t s protein

of

Subsequent addition

utiilsation

fall

coincided

with

c o n c e n t r a t i o n of f r e e amino a c i d s in t h e c u l t u r e at

Levels

in p r o t e i n c o n c e n t r a t i o n .

Growth and

2).

peak

the

crustuliniforne

Growth,

protein

cultures

into

growth of the

c e a s e d and o n l y 25Z of t h e p r o t e i n had been u t i l i s e d . of

released

the in

used

quantity

the as

of

a

sharp

solution.which free

amino

a b s e n c e of s i g n i f i c a n t

a substrate

Trace q u a n t i t i e s

increase

to

support

acid

reached a declined

quantities the

in

of

continuing

of ammonium were d e t e c t e d

in the

solution only after 56 days by which time growth of the fungus has ceased.

:~88

H twic~lt

22. -~ 20. -~gO - ®

,

i

,IlO - ..o- i

~

I

1./..

.~o~

1.0.

3G

06 O.;b /,

II

IZ

16

20

Z&

"~

:t

--~ 2.Z. •-~ 110 2,G [ --

~o~

,oo.

.¢S

IJI-

co.

.:zo

EG •

SO.

o~

/,0-

.ZO.~

O6-

30-

-'~ .(

0~-

20-

.lO U

02-

1056

e~,

7'2

'CO

r~,~ Ida,/v, !

Fig. 1

Dry w e i g h t y i e l d , erlcae nitrogen

2rowing source.

protein in

liquid

utilisation culture

and amino a c i d r e l e a s e with

protein

as

sole

by H.

exogenous

289

I

20-

16~ 12J O.~' 0.60.4~

~

~

~o

~

iT

t, 1.2-

.,~

io-

| °"0,6-

3O-

Ok,,-

20=

0.2-

108

16

26

~

40

gb

~

72

80

r,me (OWsl

Fig. 2

Dry weight yield, protein utiiisation and amino acid release by H. crustuliniforme

growing

exogenous nitrogen source.

in liquid

culture

with

protein

as sole

290

(ii)

S p e c t r u m o f amino a c i d s

Growth o f

the

solution,

fourteen

concentration

fungus led of

of lost

to

in culture the

release

which c o u l d

amino a c i d s i n c r e a s e d utilisation

concentration.

the

absent,

Analysis

AA's

over

suggested

and l y s i n e

of

of

the

that

indlvidual

two

aspartlc

after

acids

into

only 8 days.

The

w i t h t i m e , up to

of t h e amlno a c i d s r e d u c e d t h e i r levels

saaples

acid,

were b e i n g s e l e c t i v e l y

aliuo

progressively

proportional

final

o f H. c r u s t u l l n i f o r m e .

be i d e n t i f i e d

a r o u n d 56 d a y s . S u b s e q u e n t l y ,

individual

filtrate

during

glutamic

of

reduction

of

which

protein

was

acid,

arginlne,

slanine

absorbed to support continued growth of

the fungus.

DISCUSSION

In qualitative acid release growth

of

teras

the pattern

by t h e two t y p e s

both

leads

which is associated

to

of aycorrhizal

a decline

In neither

or

breakdown when g l u c o s e

rates

of protein

fungus is

with rather

carbon

present

host

that

plant,

in

the

into

the culture

p r o d u c t o f growth medium.

condition it

aeeus

Both

of azino acid release

the nature

in deteruinlng

Since in the sycorrh~zal by t h e i r

acids

The

a process

than in the ecto aycorrhizal

revealed

t o t h e f u n g u s p l a y s a key r o l e

concentration,

asmoniua a significant is

and o f amino

fungus are comparable.

of free alino

in the ericold

The s t u d y o f ~. e r i c a e

utilisation

protein

breakdown and t h e q u a n t i t i e s

h o w e v e r , Much g r e a t e r

released.

in

with the release

solution. protein

of protein

the are,

fungus.

of the carbon supply

w h e t h e r or n o t a m o n i u n fungi are norsally lost

t h a n ammoniua w i l l be t h e p r o d u c t o f p r o t e i n

likely

that

is

supplied aaino-acid

breakdown i n n a t u r e .

291

It

is evident

assimilate

from t h e s e r e s u l t s

both of the fungi r e l e a s e and

amino acids when grown on a proteinaceous

observation

is potentially

carbon n u t r i t i o n

of t h e

mycorrhizsl

of great

significance

fungus and h o s t ,

n i t r o g e n c y c l i n g in s o i l .

involving

that

substrate.

for the nitrogen

as well as for

the process

and

and of

The s t u d y i s b e i n g e x t e n d e d to o t h e r s p e c i e s of

fungi and to aseptically synthesised mycorrhizal pine

This

birch

so that the

full implication

associations

of this newly

revealed pattern of nitrogen turnover can be ascertained.

REFERENCES

BAJWA. R.. ABUARGHUB. S. and READ. D.J. (1985). The Biology of Mycorrhiza in

the

Ericaceae.

mycorrhiza]

The p r o d u c t i o n

of

proteolytic

endophyte and by m y c o r r h i z a l p l a n t s .

enzymes by

the

New P h y t o l o g i s t .

101, p 469-486.

ABUZINADAH.

R.A. and READ. D.J. (1986).

nitrogen nutrition peptides

of

and p r o t e i n s

The role of proteins

ectomycorrhizal plants. by e c t o m y c o r r h i z a l

fungi.

I.

in the

Utillsation

of

New P h y t o l o g i s t .

103. 481-493.

Langdale, A.R. and Read, D.J., 1989: S u b e t r a t e d e c o m p o s i t i o n and p r o d u c t r e l e a s e by ericoid and e o t o m y c o r r h i z a l fungi g r o w n on protein. Agric., E c o s y s t e m s Environ., 28: 285-291.