Induction and Secretion of Hydrolytic Enzymes by the Biocontrol Agent Trichoderma Harzianum

181

INDUCTION AND SECRETION TRICHODERMA HARZIANUM

OF

HYDROLYTIC

ENZYMES

BY

THE

BIOCONTROL

AGENT

R. GEREMIA, D. JACOBS*, G.H. GOLDMAN, M. VAN MONTAGU and A. HERRERA-ESTRELLA Laboratorium voor Genetica, Rijksuniversiteit Gent, K.L. Ledeganckstraat 35, B-9000, Gent (Belgium); * Rijksstation voor Plantenziekten, B-9220 Merelbeke (Belgium)

ABSTRACT The production of hydrolytic enzymes by Γ. harzianum was described many years ago. However it was never studied in detail and therefore its actual role in biocontrol never proven. In order to evaluate the role of hydrolytic exoenzymes of T. harzianum in biocontrol we studied the induction and secretion of proteases and ß(l,3)-glucanases. Among the different inducers used were: autoclaved mycelia from Botrytis cinerea, Rhizoctonia solani, Fusarium oxysporum and Τ. viride, as well as some purified cell wall components. Both B. cinerea and Τ. viride stimulated specifically the synthesis and/or secretion of an alkaline proteinase, whereas laminarin, pustulan and B. cinerea glucan failed to induce this enzyme. This proteinase has been biochemically characterized and purified to homogeneity . The level of induction of ß(l,3)-glucanase activity was highest when using cell walls from R. solani and pustulan. The linear β(1,3)-glucan laminarin induced to a lesser extent. Interestingly a neutral ß-(1,3)-glucanase was specifically induced by pustulan. The effect of the induction and inhibition of these enzymes on biocontrol is being studied. 1 INTRODUCTION The

imperfect fungus Trichoderma

harzianum

of controlling many phytopathogenic

fungi

may provide

(Chet, 1987).

an effective

whereas

method

its mechanism

of action has not been extensively studied, it has been shown that this fungus is able to secrete a variety of hydrolytic ase,

enzymes

(ß(1,3)-glucanase, chitin-

protease) (Elad et al. , 1984), and to produce toxic metabolites

al. , 1983).

In

addition,

microscopy

studies

showed

that

Γ.

(Elad et

harzianum

coils

around and finally penetrates the pathogen hyphae. A

proposed

mechanism

of

action

is

the

following:

(i) hydrolytic

attack the cell wall of the phytopathogenic fungi; (ii) T. harzianum

enzymes

penetrates

the hyphae, and (iii) finally injects antibiotics into the phytopathogen. Since

the hydrolytic

enzymes

seem

to participate

in the

initial

steps

of

biocontrol, we decided to study their regulation, selecting those which seemed more

obviously

related

to biocontrol, and purify

them with

obtaining a better insight in the mechanism of biocontrol.

the

final

aim

of

182 2 MATERIALS AND METHODS 2.1 Strains

and

media

T. harzianum (10

(IMI206040) was

spores/ml)

4

of a medium

grown

described

in Erlenmeyer previously

flasks

containing

(Del Rey

400 ml

et al. , 1979)

and

incubated at 25°C for 7 days. Glucose was used as carbon source at this stage and its concentration was maintained at 5 mg/ml throughout this growth period. After this first period of growth mycelium, was harvested and brought in the same medium but without medium

were

divided

glucose. After

into

portions

of

two days 90 ml

and

of

incubation, mycelium

10-ml

aliquots

of

the

mineral medium containing the different carbon sources were added. The sources solani

were

either

cell walls

from Botrytis

laminarin

(prepared

cinerea

(Sigma),

pustulan

(Calbiochem),

as described by Ridout

one using

carbon

Rhizoctonia

et al. , 1986),

or

glucan

(prepared as described by Dubourdieu et al. , 1978) at a

final concentration of 1 mg/ml. Two parallel control experiments were out,

and same

glucose

at

5 mg/ml

and one without

carbon

source.

carried

Autoclaved

mycelia of different phytopathogenic fungi were also used. All experiments were carried out in triplicate. Mycelia were allowed to grow for 7 days, media were collected, and enzymatic activity assayed.

2.2 Enzymatic

assays

(i) ß(l,3)-glucanase.

Aliquots

of the different media were

passed

through

PD10 columns (Pharmacia-LKB) and assayed for ß-glucanase activity using lamina­ rin as substrate. The liberation of reducing sugars was measured by the method of

Nelson

(1957)

and

Somogyi

(1952) . Protein

content

of

the

samples

was

measured with the Bio-Rad protein assay. (ii) Total protease. Elad et al.

(iii) Basic be

Hide Protein Azure was used for the assay according to

(1982). proteinase

specifically

activity.

determined

The activity of the basic proteinase could

using

succinyl-Ala-Ala-Pro-Phe-p-nitroanilide

(substrate A) (Sigma).

2.3 Isoelectric

focusing

gels

Isoelectric focusing (IEF) was carried out with the concentrated samples on Ampholine-PAG

plates

pH 3-9.5

(Pharmacia-LKB)

in

a

Multiphor

system

(LKB-

Pharmacia) according to the manufacturer's instructions. Detection

of

ß(l,3)-glucanase

described previously

isoenzymes

on

IEF

gels

was

performed

(Pan et al. , 1989) and detection of proteolytic

on IEF gels was performed according to Heeb and Gabriel (1984).

as

activity

183 2.4. Purification

of the basic

proteinase

The basic proteinase was purified from the supernatant ture. The purification procedure pitation.

The

protein

consisted

precipitating

at

of an induced cul­

of a differential

70% was

loaded

on

(NH ) SO 4

a

2

preci­

A

DEAE-Sepharose

chromatography column (Pharmacia) at pH 9 and subsequently onto a Bio-Gel (Bio-Rad) chromatography

column. After these steps, a homogeneous

was obtained as judged by Polyacrylamide gel electrophoresis

P-30

preparation

(SDS/PAGE).

3 RESULTS AND DISCUSSION 3.1 Secretion

of

ß(l,3)-glucanase

β(1,3)-glucanases zianum

are

secreted

at

relatively high basal

level by

Τ.

har­

(Table 1 ) .

TABLE 1 Glucanase activity in culture supernatants Carbon source

Glucanase activity Units (/xmol/mg protein)

None Glucose Laminarin Pustulan R. solani cell walls B. cinerea glucan

92.8 98.6 143.8 261.7 157.8 116.6

These enzymes are thought to attack B(l,3)-glucans commonly present in fungal cell walls. In order to establish whether the secretion of this enzyme is fur­ ther stimulated by degradation products of the cell wall, we grew the fungi in the

presence

pustulan

of

the

following

(β(1,6)-glucan), B.

polysaccharides:

(ß(l,3)-glucan), solani

cell walls

(Table 1 ) . The presence of laminarin as well as that of R.

solani

cell

stimulated

Surprisingly, enzyme was

pustulan,

a very

the a

production

and/or

polysaccharide

effective

induction

glucan

laminarin

(ß(l,3/1,6)-glucan) and R.

walls

cinerea

inducer.

The

presence

of a biochemically

inducer. B.

detected

in

which cinerea

these

different

secretion is

not

of a

ß(l,3)-glucanases. substrate

for

this

glucan did not behave as an

experiments

ß(l,3)-glucanase.

could

be

due

Therefore,

to

the

enzymatic

detection on IEF was used to determine the presence of isoenzymes. In the pres­ ence of glucose, there are only two neutral glucanases present (Fig. 1 ) . In the absence of glucose and the presence of the different polysaccharides, two addi­ tional acidic glucanases are found. The observed increase in the total glucan­ ase activity could be explained by the appearance of the acidic ß(l,3)-glucan­ ases. Since high protease activity was also found in the culture supernatants, it is possible that there are only one neutral and one acidic glucanase, which

184

Fig. 1. ß(l,3)-glucanases isoenzymes on IEF gels. 1, no carbon source; 2, gluc­ ose; 3, laminarin; 4, pustulan; 5, R. solani cell walls; 6, B. cinerea glucan.

are

cleaved by the protease, producing

an active

form with

a different

iso­

electric point.

3.2 Protease

secretion

In order to determine if the stimulation of the production of ß(l,3)-glucan­ ase

is a general process

total proteinases

present

involving

other

enzymes, we decided

in the supernatants

(Table 2 ) .

role of proteases was not evaluated yet, T. harzianum of sterile mycelia of different phytopathogenic agent, T. viride.

to measure

Since

the

the possible

was grown in the presence

fungi, and another biocontrol

The levels of secreted protease did not differ much with

TABLE 2 Protease activity in culture supernatants Carbon source

Total proteinase activity (OD ) 590

None Glucose Laminarin Pustulan R. solani cell walls Β. cinerea glucan R. solani B. cinerea Τ. viride F. oxysporum OD and O D determined. 5 9 0

4 0 5

,

optical

0.274 0.304 0.155 0.178 0.362 0.252 0.223 0.339 0.587 0.222 densities

Basic proteinase activity
_

0.205 2.05 2.1 0.365 at 590 run and 405 run, respectively;

-, not

185 varying inducers. But because previous experiments using Novozyme 234 revealed the presence

of different proteinases, we decided

to separate

them using

and measure the gelatin hydrolytic activity as described previously

IEF

(Heeb and

Gabriel, 1984). In this way we observed the presence of a neutral proteinase on all the supernatants analyzed

(Fig. 2 ) . Interestingly, a basic proteinase was

only observed in the culture media in which Trichoderma ence of autoclaved mycelia of B. cinerea

was grown in the pres­

and Γ. viride (Fig. 2 ) .

Fig. 2. Proteinases present in culture supernatants.

This result suggests that the induction of synthesis and/or secretion of the basic proteinase is due to a different process from that of the ß(l,3)-glucan­ ase. Since the basic proteinase was not present in cultures grown in the pres­ ence

of

different

(Table 2 ) , the

polysaccharides

factor

responsible

commonly for

such

present

in

fungal

induction

is

either

cell a

walls

different

polysaccharide (not assayed), or a chemically different compound. In view of these results, we suppose that the hydrolytic action of the constitutively

secreted

enzymes

releases a molecule

from

fungal

cell walls

that

could act as an inducer for the secretion of the acidic ß(l, 3)-glucanase. The coordinated

action

of

this

enzyme

and

proteinase) degrades more effectively

the

existing

ones

(chitinase,

neutral

the fungal cell wall and releases

other

compounds that induce the synthesis and/or secretion of the basic proteinase. This

enzyme

present

could

in the

be

responsible

cell wall,

and even

for

the

extensive

degradation

in the cell membrane,

of

giving way

protein to

the

186 penetration of the cell by

Trichoderma.

Since this basic proteinase seems to be correlated with biocontrol we went ahead with its purification.

3.3 Characteristics

of the basic

proteinase

We were able to find and purify to homogeneity a basic proteinase present in induced cultures. Its molecular mass was estimated by SDS/PAGE to be approxima­ tely 31 kDa and its pi 9.2

(Fig. 2 ) . It hydrolyzed

substrate A, specific

chymotrypsin, but no other

substrates

trypsin and elastase, and

aminopeptidase phenyl methyl

specific

for

for

substrates at very low rate. The fact that it is inhibited by sulfonyl

fluoride

(PMSF),

allowed us

to define

it as a

serine

proteinase. Substrate A was hydrolyzed only by the supernatants that contained the basic proteinase (Table 2) and this activity was inhibited by PMSF.

4 CONCLUSIONS We can conclude that (i) at least two ß(l-3)-glucanases harzianum; glueans,

(ii) the acidic ß(l,3)-glucanase including

ß(l,6)-glucans;

induced in T. harzianum

(iii)

are secreted by T.

is induced by different

a basic

proteinase

is

types of

specifically

by fungi with high protein content in their cell wall

and not induced by different cell wall glucans; and

(iv) β(1,3) -glucanase and

proteinase are most likely induced via independent mechanisms.

5 REFERENCES Chet, I, 1987. Trichoderma - application, mode of action, and potential as a biocontrol agent of soilborne plant pathogenic fungi. In: I. Chet (Editor), Innovative Approaches to Plant Disease Control, Wiley & Sons, New York, pp. 137-160. Del Rey, F., Garcia-Acha, I., and Nombela, C , 1979. The regulation of ß-glucanase synthesis in fungi and yeast. J. Gen. Microbiol., 110:83-89. Dubourdieu, D. , Fournet, B., Bertrand, Α., and Ribereau-Gayon, P., 1978. Identification du glucane secrete dans la baie du raisin par Botrytis cinerea. C R . Acad. Sei. D 286:229-231. Elad, Υ., Chet, I., and Henis, Υ., 1982. Degradation of plant pathogenic fungi by Trichoderma harzianum. Can. J. Microbiol., 28:719-725. Elad, Υ., Chet, I., Boyle, P., and Henis, Υ. , 1983. Parasitism of Trichoderma spp. on Rhizoctonia solani and Sclerotium rolfsii - scanning electron microscopy and fluorescence microscopy. Phytopathology, 73:85-88. Elad, Υ. , Barak, R. , and Chet, I., 1984. Parasitism of Sclerotium rolfsii sclerotia by Trichoderma harzianum. Soil Biol. Biochem., 16:381-386. Heeb, M.J., and Gabriel, 0., 1984. Enzyme location on gels. Meth. Enzymol., 104:416-441. Nelson, N.J., 1957. Colorimetric analysis of sugars. Meth. Enzymol., 3:85-86. Pan, S.-Q., Ye, X.-S., and Kuc, J., 1989. Direct detection of ß-1,3-glucanase isozymes on Polyacrylamide electrophoresis and isoelectrofocusing gels. Anal. Biochem., 182:136-140. Ridout, C.J., Coley-Smith, J.R., and Lynch, J.M., 1986. Enzyme activity and electrophoretic profile of extracellular protein induced in Trichoderma spp. by cell walls of Rhizoctonia solani. J. Gen. Microbiol., 132:2345-2352. Somogyi, M. (1952). Notes on sugar determination. J. Biol. Chem., 195:19-23.