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Biological Control 31 (2004) 346–352

Effects of Paecilomyces lilacinus protease and chitinase on theeggshell structures and hatching of Meloidogyne javanica juveniles

Alamgir Khana,*, Keith L. Williamsa, Helena K.M. Nevalainenb

a Proteome Systems Ltd., 1/35-41 Waterloo Road, North Ryde NSW 2113, Australiab Department of Biological Sciences, Macquarie University, NSW 2109, Australia

Received 23 February 2004; accepted 25 July 2004

Available online 24 August 2004

Abstract

A serine protease and an enzyme preparation consisting of six chitinases, previously semi-purified from a liquid culture of Paeci-

lomyces lilacinus strain 251, were applied toMeloidogyne javanica eggs to study the effect of the enzymes on eggshell structures. Trans-

mission electron microscopic studies revealed that the protease and chitinases drastically altered the eggshell structures when applied

individually or in combination. In the protease-treated eggs, the lipid layer disappeared and the chitin layer was thinner than in the

control. The eggs treated with chitinases displayed large vacuoles in the chitin layer, and the vitelline layer was split and had lost its

integrity. The major changes in the eggshell structures occurred by the combined effect of P. lilacinus protease and chitinases. The lipid

layer was destroyed; the chitin layer hydrolyzed and the vitelline layer had lost integrity. The effect ofP. lilacinus protease and chitinase

enzymes on the hatching ofM. javanica juveniles was also compared with a commercially available bacterial chitinase. The P. lilacinus

protease and chitinase enzymes, either individually or in combination, reduced hatching ofM. javanica juveniles whereas a commercial

bacterial chitinase had an enhancing effect. Some juveniles hatched when the eggs were exposed to a fungal protease and chitinase mix-

ture. We also established that P. lilacinus chitinases retained their activity in the presence of endogenous protease activity.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Paecilomyces; Protease; Chitinase; Meloidogyne; Hatching; Eggshell; Ultrastructure; Transmission electron microscopy; Proteolysis

1. Introduction

The eggshell is the toughest part of nematode eggs

and plays a role in their resistance to chemicals and bio-

logical nematicides (Wharton, 1980). The eggshell may

consist of one to five layers depending on the nematode

order. The most common structure features an inner li-

pid layer, a middle chitinous layer (major components)and an outer vitelline layer (Bird and McClure, 1976).

In the tylenchid root-knot nematode Meloidogyne java-

nica (Treub) Chitwood, the vitelline layer is very thin

(10–40 nm) and contains lipoprotein. The 400 nm thick

major chitin layer is combined with protein to form a

1049-9644/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.biocontrol.2004.07.011

* Corresponding author. Fax: +61 2 9889 1805.

E-mail address: alamgir.khan@proteomesystems.com (A. Khan).

chitin–protein complex. M. javanica eggshells contain

50% protein and 30% chitin (Bird and Bird, 1991).

The main role of the chitinous layer may be to provide

structural strength and to protect the lipid layer. The li-

pid layer consists of a series of lipoprotein membranes

(Wharton, 1980) and is thickest at the poles (0.02–

0.04 lm). The lipid layer is responsible for the imperme-

ability of some nematode eggshells, and it protects theeggs from harmful chemicals (Wharton, 1980). If the

chitinous layer is removed, the lipid layer is easily

damaged.

Nematode eggshell and cuticle are infection sites for

microorganisms applied for biological control of nema-

todes. Protease, chitinase, and lysozyme enzymes are

commonly produced and secreted by fungal antagonists

to degrade the nematode eggshell and cuticle (Bonantset al., 1995; Dackman et al., 1989; Gupta et al., 1993).

A. Khan et al. / Biological Control 31 (2004) 346–352 347

A number of studies have shown the importance of pro-

teases in this process. For example, Segers et al. (1994)

demonstrated that an egg-parasitic fungus, Pochonia

chlamydosporia (Goddard) Zare and Gams (formally

named as Verticillium chlamydosporium Goddard), se-

creted several proteases in a submerged culture thathydrolyzed proteins from the outer layer of the eggshell

of a plant-parasitic nematode Meloidogyne incognita

(Kofoid and White) Chitwood, thereby exposing the

chitin layer. Secretion of a serine protease by Verticil-

lium suchlasporium Gams and Dackman was also ob-

served during infection of Heterodera schachtii

Schmidt eggs (Lopez-Llorca and Robertson, 1992a).

Immunocytochemical localization of this protease sug-gested that the enzyme was directly involved in the infec-

tion process. Purified protease and chitinase enzymes

have also been shown to disrupt the eggs of an ani-

mal-parasitic nematode Haemonchus contortus Rud

(Mansfield et al., 1992). In studies of the plant-parasitic

nematode Meloidogyne arenaria (Neal) Chitwood, an

eggshell infected by Paecilomyces lilacinus (Thom) Sam-

son was shown to undergo a number of ultrastructuralchanges (Morgan-Jones et al., 1984). The authors found

that the vitelline layer was split into three unevenly

thickened discrete membranes, the chitin layer became

vacuolated and the lipid layer largely disappeared.

While several studies have addressed involvement of

protease enzymes purified from various fungi in the

infection of nematodes, information on the role of a

purified chitinase in the infection process has not beenavailable to date. In this work, we describe the effects

of a serine protease and chitinases isolated from a

non-paecilotoxin producing strain, P. lilacinus 251

(Khan et al., 2003a), on the eggshell structures and

hatching of juveniles of M. javanica.

2. Materials and methods

2.1. Extraction of M. javanica eggs

Mature egg masses were obtained from tomato roots

infected withM. javanica in the glasshouse (Holland and

Williams, 1996). Clean eggs were obtained from mature

egg masses as follows: about 50 egg masses were disag-

gregated in 1 ml of 0.5% (v/v) bleach (NaOCl) followinga procedure used by a number of authors (Bird and Self,

1995; Fitters and den Belder, 1993; Morgan-Jones et al.,

1983) but with a notably lower concentration of NaOCl

and shorter treatment time. Egg masses were vortexed

three times for 10 s. Eggs from the dispersed egg masses

were extracted with a pipette and pelleted at 14,000g for

30 s. The pellet was suspended in 1 ml of Milli-Ro (Mil-

lipore, Bedford, MA, USA) water and re-centrifuged asabove. This procedure was repeated three times to re-

move the NaOCl from the egg suspension. The final pel-

let was resuspended in 250 ll of Milli-Ro water and

used.

2.2. Effects of P. lilacinus protease and chitinase on M.

javanica eggs and hatching of juveniles

Protease and chitinase enzyme preparations used in

this study were from the culture supernatants of P. lilac-

inus strain 251 and had been previously semi-purified

(Khan et al., 2003b). On SDS–PAGE, the protease puri-

fied from the culture supernatant was seen as three ma-

jor bands, two of which were a truncated form of the

35 kDa serine protease. An ammonium sulfate precipi-

tated culture supernatant showed six active forms ofchitinases, which also were the most abundant proteins.

Thirty microliters of egg suspension (approximately

600–700 eggs) was aliquotted in each well of a 96-well

microtiter plate for the addition of enzymes. The treat-

ments were as follows: (T1) P. lilacinus protease; (T2)

P. lilacinus chitinases; (T3) P. lilacinus protease and

chitinases; (T4) Streptomyces griseus chitinase (Sigma,

St. Louis, MO, USA) dissolved in 50 mM sodium ace-tate pH 5.5; and (T5) water only. Each treatment was

replicated five times over time. Two hundred microliters

of protease (protein conc. 0.63 mg/ml) and chitinases

(protein conc. 2.5 mg/ml) were applied in T1 and T2,

respectively. For the treatment with an enzyme mixture

(T3), 100 ll of protease and 100 ll of chitinase were

added. T2 also contained the serine protease inhibitor

phenylmethylsulphonyl fluoride (PMSF) at the concen-tration of 1 mM to stop serine protease activity. From

our previous studies, it was observed that P. lilacinus

grown in minimal medium supplemented with chitin

produces some serine protease and adding PMSF in

the chitinase sample did not interfere with chitinase

activity. For T4 and T5, 200 ll of S. griseus chitinase

(protein conc. 1 mg/ml) or 200 ll of water were used.

A microtiter plate with all ingredients added was incu-bated at room temperature (21 ± 1 �C) for 6 days.

2.3. Light microscopic observations of protease and

chitinase treated eggs

Enzyme treated nematode eggs (described in Section

2.2) were observed with light microscopes. Hatched

and dead (motionless) juveniles were counted with a ste-reomicroscope at 3 and 6 days after incubation. Photo-

graphs of eggs were taken using an inverted microscope

after 6 days incubation before samples were prepared

for transmission electron microscopy.

2.4. Sample preparation for transmission electron

microscope

Enzyme-treated eggs were fixed using a freeze fixation

method (Holland et al., 1999). The egg samples were

348 A. Khan et al. / Biological Control 31 (2004) 346–352

drawn into microcellulose tubes (300 lm diameter) by

capillary suction. Tubing containing the eggs was then

cut into sections of about 5 mm length and plunged into

liquid propane cooled with liquid nitrogen. The samples

were transferred into the Reichert CS Auto cooler (Reic-

hert, Austria) at �85 �C. Freeze substitution was carriedout in a substitution fluid containing 0.5% (v/v) aqueous

uranyl acetate, 1% (v/v) osmium tetroxide and 2% (v/v)

gluteraldehyde in methanol at �85 �C for 72 h. After

incubation at �85 �C, the temperature was set to rise

to �30 �C over a period of 15 h followed by placing

the samples in a �20 �C freezer for infiltration with med-

ium grade LR White resin (London Resin, supplier Pro-

SciTech, Qld, Australia). The samples were infiltratedwith 1:1 resin:methanol for 1 h, pure resin 1 h and pure

resin overnight. Samples were brought to room temper-

ature, embedded in LR White resin and polymerized for

24 h at 60 �C using a Lynxel Microscopy Tissue Proces-

sor (Australian Biomedical, Vic, Australia). After poly-

merization, 1 lm sections were cut on the Reichert ultra

cut (Reichert, Austria) with glass knives and stained

with 1% (w/v) methylene blue and observed under a lightmicroscope. After cutting with glass knives, further

70 nm sections were obtained with a Drucker diamond

knife on the Reichert ultra cut. The sections were stained

with 2% (v/v) aqueous uranyl acetate for 20 min and

Reynolds lead citrate (Reynolds, 1963) for 4 min. The

stained sections were viewed and photographed with a

Philips CM 10 transmission electron microscope (Phi-

lips, The Netherlands). Three samples of each treatmentwere examined with transmission electron microscope

(TEM).

2.5. Observations on the resistance of P. lilacinus chiti-

nase enzymes to proteolysis by its own serine protease

Resistance of P. lilacinus chitinases to proteolysis by

its own serine protease was observed by measuring theactivities of the chitinases and protease in solution.

(T1) P. lilacinus protease (a serine protease); (T2) chitin-

ases with PMSF; and (T3) a mixture of both protease

and chitinases (50:50) without PMSF were left on the

lab bench at room temperature (21 ± 1 �C). Samples

were taken from these enzymes at 0, 12, 24, 48 h, and

Table 1

Effects of Paecilomyces lilacinus protease and chitinases on the hatching of M

Treatment % Hatched juveniles after

3 days incubation (±SE)

Protease (T1) 4d (±0.5)

Chitinase (T2) 14b (±0.4)

Protease and chitinase (T3) 8c (±0.4)

S. griseus Chitinase (T4) 27a (±2.1)

Water (T5) 13b (±1.0)

The treatment means were separated according to Duncan�s new multiple ra

letter are not significantly different (P = 0.05). The results were obtained from

were counted as dead. SE, standard error.

7 days after incubation and protease and chitinase activ-

ities were measured accordingly to establish the effect of

the endogenous protease on chitinase activity.

2.6. Statistical analysis

Effects of P. lilacinus protease and chitinase on the

hatching ofM. javanica juveniles experiment was carried

out as completely randomized design (CRD) with five

replications and analysis of variance (ANOVA) was car-

ried out on actual values. When the overall F test was

significant, the treatment mean values were compared

with Duncan�s new multiple range test (DNMRT) at

the 5% level of significance.

3. Results

3.1. Effects of P. lilacinus protease and chitinase on the

hatching of M. javanica juveniles

Protease and chitinase enzymes applied either indi-vidually or in combination significantly reduced

(F = 62, df = 4, 20, P = 0.01 for 3 days, and F = 595,

df = 4, 20, P = 0.01 for 6 days) the hatching of M. java-

nica juveniles (Table 1). Hatching of juveniles was lowest

(P = 0.05) when the eggs were exposed to protease (T1)

and a mixture of fungal protease and chitinase (T3).

Protease treatment reduced the hatching by 69 and

88% after incubation of 3 and 6 days, respectively, andthe reduction caused by the enzyme mixture was 38%

(3 days) and 79% (6 days) over the water-treated control

(T5). In both cases, the reduction of hatching was signif-

icant (P = 0.05). Hatching of M. javanica juveniles in

eggs treated with P. lilacinus chitinase (T2) increased

by 8% (not significant) at 3 days but was reduced by

60% at 6 days (significant, P = 0.05) over water-treated

eggs. By contrast, chitinase from S. griseus (T4) en-hanced the hatching of M. javanica juveniles by nearly

108 % in a 3-day and 21% in a 6-day incubation, com-

pared to the water-treated control (T5). As expected,

hatching of juveniles increased in the water-treated con-

trol over time. Only 9 % of the juveniles hatched in

water died, whereas mortality in the enzyme-treated

. javanica juveniles

% Hatched juveniles after

6 days incubation (±SE)

% Dead juveniles at

6 days incubation (±SE)

5e (±0.4) 70a (±2.4)

17c (±0.4) 52b (±4.9)

9d (±0.2) 78a (±1.8)

51a (±1.4) 51b (±1.8)

42b (±1.1) 9c (±1.1)

nge test (DNMRT). Means in the same column followed by the same

the five replications of each treatment. Motionless and rigid juveniles

A. Khan et al. / Biological Control 31 (2004) 346–352 349

samples (F = 98, df = 4, 20, P = 0.01) was much higher

(Table 1). Mortality of hatched M. javanica juveniles

in enzyme-treated samples (T1, T2, T3, and T4) was

about 6- to 9-fold when compared to the water-treated

control (T5). The percentage of dead juveniles between

T1 and T3, (protease and enzyme mixture), and T2and T4 (fungal and bacterial chitinase) was not signifi-

cantly different.

3.2. Light microscopic observations of protease and

chitinase treated eggs

Physiological and morphological changes of eggs and

juveniles were first observed under a stereomicroscope

Fig. 1. Light micrographs of enzyme-treated M. javanica eggs and

juveniles. (A and B) Eggs treated with P. lilacinus chitinase. UJ points

to an unhatched juvenile, VJ shows a vacuolated hatched juvenile and

DJ a disintegrating juvenile. (C and D) Eggs treated with P. lilacinus

protease and chitinase mixture; V denotes a vacuolated unhatched

juvenile, SE points to an egg where embryogenesis seemed stopped and

VJ points to a vacuolated hatched juvenile. (E) Eggs treated with S.

griseus chitinase; NE denotes a normally developing egg and DE a

deformed egg. (F and G) Water-treated control; EE denotes empty egg

shells, NE normally developing eggs and J2 a healthy second stage

juvenile hatched from the egg. Photographs were taken from a

microtiter plate using an inverted microscope without any prior

fixation of the specimens. Scale bar = 30 lm.

after 6 days incubation with enzymes, before the treated

eggs were prepared for ultrastructural studies. Photo-

graphs were taken directly from a 96-well plate in-

spected with an inverted microscope without any

fixation of the eggs and juveniles.

Eggs and juveniles from individual protease (notshown) or chitinase treatments (Figs. 1A and B) were

strongly affected with the enzyme treatment eventually

causing death. Developing juveniles in eggs did not de-

velop further and unhatched juveniles, motionless in

eggs, were assumed dead. Bodies of the juveniles,

whether in the egg or hatched, were deformed. The juve-

nile bodies disintegrated, become vacuolated and essen-

tially transparent after 6 days incubation.Major morphological changes occurred in eggs and

juveniles exposed to the protease and chitinase mixture

(Figs. 1C and D). Practically none of the early stage eggs

developed further. The developing eggs and juveniles

were vacuolated and became transparent suggesting

hydrolysis of the egg and juvenile contents. Although

S. griseus chitinase enhanced the hatching of juveniles

(Table 1), eggs in an early developmental stage were de-formed and did not develop further (Fig. 1E). Water-

treated eggs developed and hatched out normally (Figs.

1F and G).

3.3. Ultrastructure of protease and chitinase treated eggs

Protease and chitinase enzymes of the nematopha-

gous fungus P. lilacinus caused clear changes in the egg-shell layers of M. javanica. The enzyme-treated eggs

were swollen and the eggshell lost its structure as indi-

cated by light microscopy.

In protease-treated eggs, the inner lipid layer disap-

peared. The outer vitelline layer seemed intact and the

middle chitin layer thinner than in the control (Fig.

2A). Disintegration of the eggshell structure facilitated

the penetration of liquid that caused the swelling ofeggs.

Therewere large vacuoles in the chitin layer ofM. java-

nica eggs treated withP. lilacinus chitinases (Fig. 2B). The

outer vitelline layer had split and lost its integrity, which

presumably allowed access of liquid into the egg interior

leading to swelling. The inner lipid layer of the eggshell re-

mained loosely attached to the chitin layer even after 6

days incubation with P. lilacinus chitinases.The major changes in the eggshell structures occurred

by the combined effect of P. lilacinus protease and chiti-

nase enzymes. In the combined treatment, the lipid layer

was destroyed and the thickest chitin layer extensively

hydrolyzed although the connection between the vitel-

line and chitin layers was still discernible (Fig. 2C). As

the vitelline layer was split and the chitin layer hydro-

lyzed, the eggs had lost their permeability and structuralstrength, which caused deformity and swelling. In the

water-treated eggs (Fig. 2D), all eggshell layers were eas-

Fig. 2. TEM micrographs of P. lilacinus enzymes-treated M. javanica

eggs. (A) Effect of protease treatment on the eggshell structures. The

inner lipid layer containing lipoprotein has disappeared facilitating

modification of the chitin layer. (B) Eggs treated with chitinase. The

thickest middle chitin layer has disintegrated and appears vacuolated.

The outer vitelline layer is also severely affected. (C) Combined effect

of protease and chitinase on the eggshell structures. The inner lipid

layer has been completely digested. The middle chitin layer is

disintegrated and largely digested. The outer vitelline layer has lost

integrity, but still remains attached with the middle chitin layer. (D)

Water-treated control showing intact inner lipid, middle chitin and

outer vitelline layers. Scale bar = 0.2 lm.

350 A. Khan et al. / Biological Control 31 (2004) 346–352

ily distinguishable and had the thickness described for

untreated M. javanica eggs by Bird and Bird (1991).

3.4. Resistance of P. lilacinus chitinases to degradation by

an endogenous serine protease

Both protease and chitinase enzymes remained active

in the presence of both enzymes in a mixture. Chitinase

retains its activity in the presence of serine protease

activity up to at least 7 days at room temperature(Table 2).

Table 2

Resistance of Paecilomyces lilacinus chitinases to proteolysis by an endogeno

Enzyme Activity tested 0 h

Protease (T1) Protease only 54.74

Chitinase (T2) Chitinase only 0.18

Protease and Chitinase Protease 51.35

mixture (T3) Chitinase 0.10

The experiment was conducted at room temperature (21 ± 1 �C) and the e

incubation. Protease activity was assayed using azocasein (Sigma, USA) as th

assayed using 4-methyumbelliferyl-N-acetyl-b-DD-glucosaminide (Sigma, St. L

(1987).

4. Discussion

Nematophagous fungi penetrate the host cell by

mechanical force and by producing various lytic en-

zymes (Dackman et al., 1989; Morgan-Jones et al.,

1984; Stirling, 1991). In this study, the serine proteasesecreted by P. lilacinus strain 251 stopped the develop-

ment of M. javanica eggs (Fig. 1), reduced the number

of hatching juveniles, and killed a large number of

hatched juveniles (Table 1). Similar results have been

observed by Bonants et al. (1995), who found with an-

other strain of P. lilacinus (CBS 143.75) that protease

treatment altered the embryogenesis of the eggs of

M. incognita, essentially stopping the hatching of juve-niles. Our results suggest that the P. lilacinus 251 prote-

ase, at the concentration used in this work, is

detrimental to nematode egg embryogenesis. The major

structural changes that occurred in protease and chiti-

nase treated eggshell were loss of the lipid layer and dis-

integration of the vitelline layer (Fig. 2C). Both lipid and

vitelline layers contain proteins (Bird and Bird, 1991;

Wharton, 1980) that may become digested. Vitellineand lipid layers support eggs by providing structural

uniformity and permeability characteristics, respec-

tively. Damage to these two layers caused by enzyme

treatment enabled other metabolites to permeate the

eggs causing changes such as swelling of eggs.

In eggs treated with the S. griseus chitinase, hatching

of M. javanica juveniles was enhanced (Table 1). This

agreed with results obtained by Reiss et al. (1998) whosuggested that hatching of root-knot nematode juveniles

was triggered by chitinase. Although hatching of juve-

niles was enhanced, early stage eggs were deformed

(Fig. 1E). In the hatching experiment, the age of all eggs

was not known, therefore, it is possible that the en-

hanced hatching with S. griseus chitinase was caused

by an early hatching of late stage eggs that contained

premature juveniles. The early hatched premature juve-niles may have died after hatching. In contrast, P. lilac-

inus chitinase treatment did not increase the hatching of

juveniles. This might be because the P. lilacinus chitinase

preparation contained several chitinase activities, while

S. griseus chitinase was a pure chitinase (N-acetyl-b-DD-glucosaminidase). A chitinase complex could be deleteri-

us serine protease at various time points

12 h 24 h 48 h 7 days

53.62 54.40 54.40 30.67

0.09 0.10 0.09 0.09

51.01 53.66 50.17 34.16

0.12 0.10 0.08 0.08

nzyme activities were tested at 0, 12, 24, and 48 h and 7 days after

e substrate as described by Lovrien et al. (1985). Chitinase activity was

ouis, MO, USA) as the substrate as described by O�Brien and Colwell

A. Khan et al. / Biological Control 31 (2004) 346–352 351

ous to the embryogenesis of eggs. However, this result is

comparable with the chitinase produced by a soil bacte-

rium Paenibacillus illinoisensis Shida et al. that inhibited

the hatching of M. incognita juveniles (Woo-Jin et al.,

2002).

Structural changes in the nematode eggshell were ob-served only with the P. lilacinus chitinases. The major

changes occurred in the chitin and vitelline layers (Fig.

2B), the lipid layer remained attached to the chitin layer

as in the water-treated control. As also observed with an

Alabama isolate of P. lilacinus, the chitin layer became

full of vacuoles during fungal penetration of the eggs

(Morgan-Jones et al., 1984).

The combined effect of protease and chitinases onthe eggshell structures was drastic. The lipid layer dis-

appeared and the chitin layer was reduced significantly

(Fig. 2C). The development of eggs and hatching of

M. javanica juveniles was stopped by the protease

and chitinases secreted by P. lilacinus. This result

was expected as it has been reported that protease pre-

vents egg development (Bonants et al., 1995). At the

same time, chitinases degraded the thickest chitin layerand caused splitting of the outer vitelline layer. How-

ever, since P. lilacinus protease alone affected the lipid

layer, it suggests that the enzyme gets in to the eggs

without assistance from chitinases. Partial disintegra-

tion of the vitelline and chitin layers by protease and

chitinases increased permeability, thus facilitating in-

ward flow of other metabolites, which eventually

swelled the eggs and had an adverse effect on embryo-genesis. Similar results have been obtained by Morgan-

Jones et al. (1983) with another nematophagous fungus

P. chlamydosporia (V. chlamydosporium) infecting M.

arenaria eggs. The permeability of eggshells may be di-

rectly related to the presence or absence of a lipid

layer, which is thought to consist of lipoproteins (Bird,

1971). Where a protease was present, the lipid layer

was removed suggesting cleavage of lipoproteins byprotease.

The published literature suggests that the rupturing

of eggshell layers of cyst nematodes during fungal pene-

tration occurs by means of mechanical pressure as well

as enzymatic digestion (Lopez-Llorca and Robertson,

1992b; Stirling, 1991). Results from our work clearly

showed that the disintegration of vitelline, chitin and li-

pid layers ofM. javanica eggshells could be caused solelyby enzymatic degradation by P. lilacinus protease and

chitinase. Therefore, enzymes may facilitate physical

penetration and improve efficiency of infection.

For effective penetration of nematode eggs, nemato-

phagous fungi must produce protease and chitinase en-

zymes at the same time to degrade different eggshell

layers. In some earlier studies with bacteria, chitinases

were degraded because of proteolysis by endogenousprotease (Blaak and Schrempf, 1995; Hiroshi et al.,

1999; Romaguera et al., 1992). In our study, chitinase

activity was retained for 7 days in a mixture also con-

taining protease; therefore, endogenous chitinases of

P. lilacinus strain 251 are not sensitive to degradation

by endogenous serine protease.

Acknowledgments

We thank the Microscopy unit of the Department of

Biological Sciences, Macquarie University. Special

thanks are due to Debra Birch and Vienny Brown for

assistance in transmission electron microscopy and

photo printing. This work was funded by the Australian

Post-graduate Award to Alamgir Khan.

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