ORIGINAL PAPER

Hydrolysis of fungal and plant cell walls by enzymatic complexesfrom cultures of Fusarium isolates with different aggressivenessto rye (Secale cereale)

Jolanta Jaroszuk-Sciseł • Ewa Kurek

Received: 22 December 2011 / Revised: 25 January 2012 / Accepted: 6 February 2012 / Published online: 3 March 2012

� Springer-Verlag 2012

Abstract The efficiency of hydrolysis of fungal (Fusar-

ium spp.) cell wall and rye root cell wall by crude enzy-

matic complexes from (42-day-old) cultures of three

F. culmorum isolates, a plant growth–promoting rhizosphere

isolate (PGPF) DEMFc2, a deleterious rhizosphere isolate

(DRMO) DEMFc5, and a pathogenic isolate DEMFc37, as

well as two other, pathogenic isolates belonging to

F. oxysporum and F. graminearum species was studied. In the

enzymatic complexes originating from the Fusarium spp.

cultures, the activities of the following cell wall–degrading

enzymes were identified: glucanases, chitinases, xylanases,

endocellulases, exocellulases, pectinases, and polygalac-

turonases. The preparation originating from a culture of the

PGPF isolate was the least efficient in plant cell wall

(PCW) hydrolysis. There were no significant differences in

the efficiency of PCW hydrolysis between preparations

from cultures of the DRMO and the pathogenic isolates.

PGPF was the most efficient in liberating reducing sugars

and N-acetylglucosamine (GlcNAc) from fungal cell walls

(FCW). Xylanase activities of the enzymatic complexes

were strongly positively (R [ ?0.9) correlated with their

efficiency in hydrolyzing PCW, whereas chitinase activi-

ties were correlated with the efficiency in FCW hydrolysis.

Keywords Efficiency of cell wall hydrolysis � PGPF �DRMO � Pathogenic fungi � Xylanase activity �Chitinase activity

Introduction

The activities of lytic enzymes hydrolyzing cell wall

polysaccharides (CWDEs) of fungi as well as plants

increase substantially in aging culture filtrate of filamen-

tous fungi (Alfonso et al. 1995b; Douaiher et al. 2007;

Jaroszuk-Sciseł et al. 2011).

The lytic enzymes released from microorganisms could

be involved in the control of fungal growth under specific

conditions via various mechanisms (Boothby and Magreola

1984). Fungal cell wall (FCW) degrading enzymes are

probably involved in the penetration and infection of plants

(Annis and Goodwin 1997; Keon et al. 1987). CWDE

secreted by a pathogen may diffuse in the apoplast and

react with host cell wall constituents, thereby facilitating

pathogen penetration through the loosened host cell walls

and middle lamella matrices (Annis and Goodwin 1997;

Carpita and Gibeaut 1993). CWDE synthesized during

plant infection by F. culmorum (Kang and Buchenauer

2000) include cellulases, xylanases, and pectinases.

The plant cell wall (PCW) is thought to be a dynamic

and metabolically active structure that functions as a

potential barrier to the penetration and spread of pathogens.

It is at the same time, a substrate for extracellular enzymes

secreted by pathogens (Akimitsu et al. 2004; Annis and

Goodwin 1997) and a reservoir of signal molecules regu-

lating or activating plant defense responses to microbial

attack (Degefu et al. 1995; Hatsch et al. 2006). Fungal

glycosidases such as b-galactosidases, xylosidases, and

arabinosidases release sugar moieties that can be used by a

Communicated by Erko Stackebrandt.

J. Jaroszuk-Sciseł (&) � E. Kurek

Department of Environmental Microbiology,

Institute of Microbiology and Biotechnology,

Maria Curie-Skłodowska University,

Akademicka St. 19, 20-033 Lublin, Poland

e-mail: jolanta.jaroszuk-scisel@poczta.umcs.lublin.pl

E. Kurek

e-mail: e.kurek@poczta.umcs.lublin.pl

123

Arch Microbiol (2012) 194:653–665

DOI 10.1007/s00203-012-0803-4

fungus as a nutritional source during its growth through

plant tissues (Akimitsu et al. 2004). It has been demon-

strated that cereal pathogens produce more xylanase

activity than other cell wall–degrading enzymes that use

cereal cell walls as a C source (Apel et al. 1993; Degefu

et al. 2001; Giesbert et al. 1998; Lalaoui et al. 2000; Phalip

et al. 2009; Wanjiru et al. 2002; Wu et al. 1995, 2006). In

graminaceous crops and grasses, arabino-xylan accounts

for up to 40% of the walls (Belien et al. 2006; Carpita

1996; Hatsch et al. 2006; Labavitch and Ray 1978).

Earlier studies (Jaroszuk-Sciseł et al. 2008) on three

Fusarium culmorum isolates originating from the rhizo-

sphere of cereal plants indicated that their interactions with

rye plants were quite different from one another. Inocula-

tion of seedlings with isolate DEMFc2 resulted in a 20%

increase in shoot fresh weight, whereas DEMFc5 and

DEMFc37 caused more than a 20% and a 38% reduction in

this parameter and fusariosis symptoms in 14-day-old

plants, respectively. The two rhizosphere isolates (DEM-

Fc2 and DEMFc5) colonized the epidermis and the cortex

but were not found in vessels, while the pathogen colonized

all three layers of root cells. The number of DEMFc37

colony forming units (CFU) isolated from plant tissues was

much higher than for the rhizosphere isolates in spite of the

same number of macroconidia used as inoculum. The CFU

number of the pathogenic isolate originating from the

interior of roots of plants pre-colonized with the rhizo-

spheric isolates was as low as 10% of the number of

pathogenic strain CFU isolated from plants inoculated with

the pathogen alone. A study of in vitro interactions

between the rhizosphere isolates and the pathogen suggests

that changes in plant colonization by the pathogen

observed in the presence of the rhizosphere isolates were

not connected with inhibition of the growth of the former

by a direct action of the latter.

Cytological studies of root sections 4 days after inocu-

lation indicated the presence of wall appositions, which are

symptoms of a defense reaction in plant cells, associated with

a fungal infection (Jaroszuk-Sciseł et al. 2008). This obser-

vation suggested that the 24-h interval between inoculation

with a rhizosphere strain and the introduction of the pathogen

may be sufficient to start a cascade of plant reactions

involved in the induced systemic reaction. Probably, chitin

oligosaccharides released by CWDE activities could be

effective elicitors of plant resistance responses (Furman-

Matarasso et al. 1999; Montesano et al. 2003).

Recently, complexes of CWDE have been found in

supernatants of autolyzing cultures of F. culmorum strains,

and the activities of particular enzymes have been deter-

mined (Jaroszuk-Sciseł et al. 2011).

The aim of the present study was to find answer whether

the efficiency of degradation of cell walls of plants (rye

roots) and fungi by enzymatic complexes originating from

supernatants of rhizosphere (plant growth promoting

[PGPF] and deleterious [DRMO]) and pathogenic Fusar-

ium spp. cultures could be related to differences in the type

of fungus–plant interactions.

Materials and methods

Fusarium spp. isolates

Five Fusarium spp. isolates belonging to three species were

used in this study: three Fusarium culmorum (W.G. Smith)

isolates with distinct effects on rye growth—two non-

pathogenic rhizospheric isolates (DEMFc2 and DEMFc5)

and one pathogenic isolate (DEMFc37) (Jaroszuk-Sciseł

et al. 2008), as well as two additional pathogenic isolates

provoking fusarium wilt symptoms and a 30% reduction in

shoot fresh weight—a Fusarium graminearum Schwabe

(teleomorph: Gibberella zeae Schweinitz) isolate (DEM-

Fc36) (Kurek and Jaroszuk 1997) and a pathogenic

Fusarium oxysporum Schlechtendal isolate (DEMFc38)

(Wolska-Mitaszko et al. 2007). All these Fusarium spp.

isolates are deposited at the Department of Environmental

Microbiology’s Fungal Collection (DEM), Maria Curie-

Skłodowska University in Lublin, Poland, and in Centra-

albureau voor Schimmelcultures Collections (CBS)

(accessed 16/07/06), P.O. Box 85167, NL-3508 AD

Utrecht, The Netherlands, http://www.cbs.knaw.nl/database/

index.htm. Nucleotide sequences of the isolates were depos-

ited in the NCBI GenBank.

The two nonpathogenic rhizosphere isolates, the PGPF

isolate DEMFc2 (CBS 120098, NCBI accession number

for nucleotide sequence DQ453700) and the DRMO isolate

DEMFc5 (CBS 120101, DQ450880), had been isolated

from the rhizosphere of healthy rye (Secale cereale L.)

grown in a field in the Lublin region (lat. 51.1500N and

long. 22.3400E) in Poland at the tillering stage (March) of

rye growth. The pathogenic isolates F. culmorum DEM-

Fc37 (CBS 120103, DQ450878), F. graminearum DEM-

Fc36 (CBS 120102, DQ453701), and F. oxysporum

DEMFc38 (CBS 120104, DQ450879) had been isolated

from winter wheat (Triticum aestivum L.) plants with

severe fusariosis symptoms.

The isolates were stored on rose bengal and strepto-

mycin (Martin 1950) 2% agar slants [(glucose, 10.0 g;

proteose peptone, 5.0 g; KH2PO4, 1.0 g; MgSO4, 0.5 g;

agar, 15.0 g; streptomycin, 30.0 mg; rose bengal, 30.0 mg

in 1,000 mL of H2O distilled water (DW)] at 4�C.

Preparation of macroconidia

Macroconidia for the preparation of inocula were obtained

from a culture grown on Martin liquid medium without

654 Arch Microbiol (2012) 194:653–665

123

rose bengal dye and were prepared as described by

Jaroszuk-Sciseł et al. (2008, 2009). The isolates were culti-

vated in the darkness at 20�C and 60% of relative humidity

in an Innova 4900 growth chamber (New Brunswick

Scientific Co Inc., Edison, NJ, USA) at 120 rpm for 7 days,

and then, the cultures were filtered through 5 layers of

sterile gauze (cotton). Supernatants were collected, and the

macroconidia were pelleted by centrifugation (Beckman

J2-HS) at 10,000g for 15 min. The macroconidia were

washed three times by suspension in sterile DW (SDW)

and vortexed vigorously. Then, the numbers of macroco-

nidia in suspension were determined in a hemocytometer,

using an LM Nikon Eclipse E200 light microscope, and

adjusted by dilution to the desired concentration (density).

Preparation of mycelium

Mycelium biomass was obtained from 5-day cultures of the

Fusarium spp. isolates in 250 mL liquid Martin medium

without rose bengal dye in 1,000-mL Erlenmeyer flasks

shaken at 120 rpm in an Innova 4900 growth chamber. Liquid

Martin medium was inoculated with 1 9 105 CFU mL-1 of

macroconidia. The obtained biomass was separated by cen-

trifugation at 10,0009g and washed with SDW. The centri-

fugation was repeated five times. Next, the fungal mycelium

was frozen in liquid nitrogen, dried by lyophilization using

FreeZone 6 l (Labconco Co, Kansas City, Missouri, USA),

and stored for use in the preparation of FCW material.

Preparation of fungal cell walls (FCW)

Lyophilized five-day-old mycelia were used for the prep-

aration of FCW material according to the procedure

described by Gomez-Miranda et al. (1990) and recom-

mended by Alfonso et al. (1992). The mycelia were dis-

rupted at 4�C in an Ultra-Turax Ika�-Werke T18

homogenizer at 16,000 rpm for 10 min at 1-min intervals

and then centrifuged (Beckman J2-HS) at 10,0009g. The

pellet was frozen in liquid nitrogen, ground in a mortar, and

washed 5 times by centrifugation with SDW. Then, the

fungal mycelia were disintegrated by ultrasonic (Sonicator

XL2020, Misonix Inc, New Yersey, USA) treatment (15

intervals per 1 min at 150 W) and washed by centrifuga-

tion (10 min at 10,0009g) alternately with phosphate

buffer (50 mM, pH 6.5) and NaCl (1 M). This process was

repeated until the cell walls were free of cytoplasmic

material (protein-free), as seen under the light microscope

after Coomassie staining (i.e., until disappearance of

absorbance at 260–280 nm). Next, the cell walls were

washed ten times with SDW to eliminate NaCl. The

obtained FCW were dried by lyophilization and stored at

4�C. The FCW were (1) added to the medium as a carbon

source, (2) used as a substrate to determine the ability of

crude enzymatic preparations to hydrolyze FCW, and (3)

used in the next step to obtain FCW fractions.

Fractionation of FCW fractions

Fungal cell walls fractions were obtained using the chem-

ical method (Alfonso et al. 1995b; Gomez-Miranda et al.

1984, 1990; Leal et al. 1992) of extraction with 1 N NaOH

(Fig. 1). The following fractions were obtained: (a) (F1S)

galacto-manno-glucan (alkali soluble and water soluble at

20�C); (b) (F1I) glucan weakly associated with chitin,

precipitated with ethanol (alkali soluble at 20�C and water

insoluble); (c) (F3) glucan strongly associated with chitin

(alkali soluble at 70�C and water insoluble, obtained after

precipitation with ethanol; and (d) (F4) glucan-chitin

(alkali insoluble at 70�C and water insoluble).

Plant material

Seeds of rye (Secale cereale L. cv Dankowskie Złote pur-

chased from ‘‘Danko’’ Company Choryn–Racot, Poland)

were used in the experiments. They were surface-sterilized

according to the following procedure: the seeds were washed

in tap water for 30 min, then soaked for 10 min in a solution

of HgCl2 (0.1%, w/v), and next soaked for 10 min in a

solution of H2O2 (30%, v/v). After these treatments, they

were rinsed ten times in SDW. The seeds were then trans-

ferred into sterile Petri dishes (25 seeds per dish) and soaked

with 2 mL SDW and subsequently germinated for 5 days in

the darkness at 20�C and 60% of relative humidity in an

Innova 4900 growth chamber. Rye roots were separated from

the tops. The roots were five times washed with SDW with

agitation at 150 rpm, frozen in liquid nitrogen, lyophilized,

and stored at 4�C for use in the preparation of PCW.

Preparation of PCW

Protein- and lipid-free PCW was obtained from roots of

5-day-old rye seedlings (Cohen et al. 1998). The roots were

placed in a mixture of methanol and chloroform (2:1, v/v),

mixed at 150 rpm for 3 days at room temperature, and then

washed 10 times with SDW during 1 day. In the next step,

PCW was obtained using the method described above for

the preparation of FCW. PCW was used (1) in culture

medium as a carbon source or (2) as a substrate for the

determination of the ability of crude enzymatic prepara-

tions to hydrolyze PCW.

Crude enzyme preparations used to degrade PCW

and FCW

Crude enzyme preparations were obtained from 42-day-old

autolyzing culture filtrate liquids of Fusarium spp.

Arch Microbiol (2012) 194:653–665 655

123

cultivated in 1,000 mL Erlenmeyer flasks with 200 mL of

modified liquid synthetic Reyes and Byrde (1973) medium

[KH2PO4, 1.0 g; MgSO4 � 7H2O, 0.5 g; KCl, 0.5 g;

(NH4)2SO4, 0.5 g; 1.0 mL of microelements solution

(Na2B4O7 � 10H2O, 100.0 mg; CuSO4 � 5H2O, 10.0 mg;

FeSO4 � 7H2O, 50.0 mg; MnSO4 � 5H2O, 10.8 mg;

(NH4)6Mo7O24 � 4H2O, 10.0 mg; ZnSO4 � 7H2O, 70.0 mg

in 1,000.0 mL of DW) and streptomycin, 30.0 mg in

1,000.0 mL of DW] supplemented with 0.5 mg PCW or

FCW per mL (0.05%, w/v) as described by Jaroszuk-Sciseł

et al. (2011). After inoculation with 100 lL of macroco-

nidia suspensions (2 9 108 macroconidia per mL), the

fungi were cultivated in the dark at 20�C and 60% relative

humidity in an Innova 4900 growth chamber under

stationary conditions for 42 days. The fungal biomass was

separated by filtration through sterile filter paper and the

filtrate liquids were additionally centrifuged (10,0009g for

10 min). Then, the supernatants were dialyzed three times

for 24 h against distilled water using dialysis tubing with a

3.5 kDa limit, dried by lyophilization, stored at -20�C and

used as crude enzyme preparations to study the efficiency

of PCW and FCW hydrolysis. The enzyme activities of the

crude enzyme preparations were periodically predeter-

mined and found to be stable for several months. Imme-

diately before their use in cell wall hydrolysis experiments

fivefold concentrated crude enzyme preparations were

obtained by solution of lyophilized culture supernatants

powders (7.5 mg mL-1) in phosphate (0.06 M) buffers

Cell wall material (freeze-dry lyophylizate)

Extraction with 1N NaOH at 200C

Supernatant Insoluble residue

Extraction with 1N NaOH at 700C

Centrifugation Centrifugation

Dialysis against water

Precipitation with EtOH (v/v)

Supernatant Precipitate Supernatant Insoluble residue

-(1,5)- galacto-manno-

glucan

F1S

fraction alkali (20oC) soluble,

water soluble

/ -(1,3)- glucan

weakly associated with chitin

F1I

fraction alkali (20oC) soluble,

water insoluble

F3

-(1,3)- glucan

strongly associated with chitin

fraction alkali (70oC) soluble,

water insoluble

F4

-(1,3)- glucan-chitin

complex

fraction alkali (70oC)

insoluble, water insoluble

Freeze-drying lyophylization

Freeze-drying lyophylization

Freeze-drying lyophylization

Freeze-drying lyophylization

Dialysis against water

Dialysis against water

Precipitation with EtOH (v/v)

βα /βαβ β

Fig. 1 Fractionation procedure of cell wall material (according to Gomez-Miranda et al. 1984, 1990; Alfonso et al. 1995b)

656 Arch Microbiol (2012) 194:653–665

123

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Arch Microbiol (2012) 194:653–665 657

123

(pH 5.6 or pH 8.8) with 0.02% (w/v) sodium azide. These

solutions were then centrifuged for 5 min at 14,0009g and

sterilized by filtration through a 0.22 lm filter (FP 030/3

Sigma Aldrich, St. Louis, Missouri, USA).

The suspensions of the crude enzyme preparations were

used to determine the activities of b-1,3-glucanase (EC

3.2.1.6), chitinase (EC 3.2.1.14), xylanase (EC 3.2.1.8),

endocellulase (EC 3.2.1.4), exocellulase (EC 3.2.1.91),

pectinase (EC 3.2.1.15), and polygalacturonase (EC

3.2.1.15) (Table 1a, b) by methods described in detail by

Jaroszuk-Sciseł et al. (2011).

The activities of acidic and alkaline b-1,3-glucanase,

xylanase, endocellulase, exocellulase, polygalacturonase, and

pectinase were assayed according to the Alfonso et al. (1992)

method by estimating the liberation of reducing substances

during hydrolysis of a 0.5% (w/v) solution of laminarin from

Laminaria digitata (L-9634 Sigma Aldrich, St. Louis,

Missouri, USA); a 1% (w/v) suspension of xylan from oat spelts

(Sigma X-0627); a 0.7% (w/v) solution of carboxymethyl

cellulose-CMC (Sigma C-5678); a 1% (w/v) suspension of

Avicel/Sigmacell� type20 cellulose (Sigma S-3504); a 0.1%

(w/v) solution of polygalacturonic acid (Sigma 81325); and a

0.1% (w/v) solution of pectin from apple (Sigma P-8471),

respectively. The reacting mixtures for these activity assays

contained 1 mL solutions of appropriate crude enzyme

preparations and 1 mL of appropriate substrate solutions or

substrate suspensions in phosphate buffers (pH 5.6 or 8.8).

Supernatants after thermal inactivation (10 min in 100�C)

were used as controls. The reacting mixtures were incubated at

37�C for 3 h with gentle agitation (80 rpm). Immediately after

incubation, soluble reducing sugars released from these sub-

strates were measured by the Nelson (1944) and Somogyi

(1945) method modified by Hope and Burns (1987). Absor-

bance was measured at 520 nm within 20 min. For pectinase

and polygalacturonase activity measurement, D-galacturonic

acid (Fluka 73960, St. Louis, Missouri, USA) was used as a

standard and for the measurements of the other enzyme

activities glucose was the standard. Enzyme activities

expressed as U mL-1 of supernatant were calculated from the

linear portion of the activity curves. One unit of activity was

defined as the amount causing the liberation of 1 lg of

reducing sugars (glucose or galacturonic acid) equivalent

min-1 under the above-mentioned conditions and at a corre-

sponding pH.

The activities of acidic and alkaline chitinases were

determined according to the Rossner (1991) method rec-

ommended by Alef and Nannipieri (1995). Chitinase

activity was assayed by estimating the N-acetyl-D-gluco-

samine (GlcNAc) equivalents liberated after hydrolyzing

the colloidal chitin suspension. The reacting mixture was

composed of 1 mL solutions of lyophilized culture super-

natants in phosphate buffers (pH 5.6 or 8.8) and 1 mL of

1% (w/v) colloidal chitin suspension in phosphate buffers

(pH 5.6 or 8.8). Thermally inactivated (10 min in 100�C)

supernatant was a control. These reacting mixtures were

incubated at 37�C for 3 h with gentle agitation (80 rpm).

The reaction was terminated by GlcNAc extraction with

2 M KCl, and then the reacting mixture was filtered. Glc-

NAc was determined in a reaction with saturated sodium

borate and p-dimethylaminobenzaldehyde (DMBA).

Absorbance was measured at 585 nm within 20 min. The

standard was Sigma A-8625 GlcNAc. Chitinase activity

expressed as U mL-1 of supernatant was calculated from

the linear portion of the activity curves. One unit of activity

was defined as the amount causing the liberation of 1 lg of

GlcNAc equivalent from chitin min-1 under the above-

mentioned conditions and at a corresponding pH.

Hydrolysis of FCW and PCW by crude enzyme

preparations

FCW and PCW degradation was studied according to the

method described by Alfonso et al. (1992). The PCW and

FCW suspensions (2 mg mL-1) in phosphate (0.06 M)

buffers (pH 5.6 or pH 8.8) with 0.02% (w/v) sodium azide

were prepared by ultrasonic disintegration (15 intervals per

1 min at 150 W) and then sterilized by autoclaving and

sterile washing with SDW by centrifugation (10 min at

10,0009g). Two mL of this suspension was incubated with

2 mL of an appropriate crude enzyme preparation solution

(7.5 mg mL-1) at 37�C during 4 days. After 24, 48, 72, and

96 h of incubation, the reacting mixtures were filtered and

additionally centrifuged. Immediately after filtration and

centrifugation, liberation of reducing sugars during PCW

and FCW hydrolysis was measured using methods descri-

bed by Nelson (1944) and Somogyi (1945), and GlcNAc

liberation during FCW hydrolysis was measured according

to Rodriguez-Kabana et al. (1983) and Rossner (1991).

Appropriate controls were included: (1) the crude enzyme

preparation solution after thermal inactivation (10 min in

100�C) and (2) PCW and FCW suspensions.

Statistical analysis

Statistical analysis of data was performed on three repli-

cates from each treatment. Standard deviations (shown as

deviation bars) were determined using Microsoft� Excel

2000 (Microsoft Corp., Redmond, Washington, USA)

Analysis of variance ANOVA was used to determine sig-

nificance of differences between the individual enzyme

activities in the crude enzymatic preparation. Data were

analyzed using STATISTICA 8.0 (StatSoft Inc., Tulsa,

Oklahoma., USA) (Armitage and Berry 1987). Mean sep-

aration was accomplished using the least significant dif-

ference (LSD) test. Significance was evaluated at p \ 0.05

for all tests.

658 Arch Microbiol (2012) 194:653–665

123

The Pearson correlation coefficient (R) and the linear

regression coefficient (R2) were determined (using Micro-

soft� Excel 2000) to show the direction and strength of the

relationship between the activities of the particular

enzymes in crude preparations from the cultures of

Fusarium spp. isolates and the efficiency of these prepa-

rations in cell wall hydrolysis. The efficiency was expres-

sed as the amounts of reducing sugars released from PCW

and the amounts of reducing sugars and GlcNAc released

from FCW.

Results and discussion

Activities of CWDE in culture supernatants of F. culmo-

rum isolates grown in media with PCW or FCW as C

sources

Lytic enzymes, with activities hydrolyzing the polymers

building PCW and FCW, released by fungi as a result of

autolysis of their mycelia and secretion during their growth,

are involved both in the colonization and penetration of plant

tissues by pathogenic and endophytic fungi and in the lib-

eration of plant defense elicitors (Ebel and Casio 1994;

Montesano et al. 2003; Shibuya and Minami 2001).

For hydrolysis of PCW as well as FCW to be efficient

and complete, synergistic action of several enzymes is

required (Alfonso et al. 1995a; Collins et al. 2005; Lalaoui

et al. 2000; Perez-Leblic et al. 1982; Reyes et al. 1977).

The activities of the hydrolases present in the crude

preparations obtained from 42-day culture supernatants of

Fusarium isolates grown in a medium containing purified

rye root cell walls (PCW) and in a medium containing

purified Fusarium isolate own cell walls (FCW) as C

sources are shown in Table 1a, and b, respectively.

Activities of seven CWDE (glucanases, chitinases, xylan-

ases, endocellulases, exocellulases, pectinases, and poli-

galacturonases) were detected in solutions of crude

enzymes preparations originated from autolyzing 42-day

cultures of three Fusarium culmorum isolates differently

affecting rye growth as well as in preparations obtained

from cultures of F. graminearum and F. oxysporum. The

activities of glucanases, chitinases, xylanases, and pectin-

ases measured in those solutions were higher than those of

the other enzymes tested and were also higher in acidic

reacting mixtures than in alkaline ones.

These enzyme activities in 14-, 28- and 42-day-old

culture supernatants of these three F. culmorum grown in

media containing one of the four C sources (PCW, FCW,

glucose, and chitin) were described in the previous Jar-

oszuk-Sciseł et al. (2011) paper; however, the values from

one period only (maximal from those three periods) were

presented. The results of those earlier studies indicate that

these 42-day-old autolyzing cultures are a very good source

of CWDE enzymes, and a majority of the enzymatic

activities determined were maximal exactly on day 42.

Alfonso et al. (1995a, b) have obtained similar results

(describing maximal autolysis in 60-day cultures).

A comparison of CWDE activities in solutions of crude

ezymes preparations originated from supernatants of cul-

tures containing PCW and FCW as C sources showed that

only the activity of polygalacturonases was enhanced in all

of the cultures tested when FCW was replaced with PCW.

In the preparations obtained from supernatant of PGPF

F. culmorum DEMFc2 grown in a culture medium containing

FCW as a C source, the highest activities were measured for

acidic glucanases and chitinases. However, the activity of

glucanases was higher in the preparations originated from

culture supernatant of this isolate grown in the medium con-

taining PCW than in the medium containing FCW as a C

source (Table 1a, b).

The activity of acidic xylanase in the solutions of crude

enzymes preparations originated from cultures of DRMO

DEMFc5 and pathogenic DEMFc37 was significantly

higher when the isolates were grown in the medium con-

taining PCW as a C source than when they were grown in

the presence of FCW as a C source.

The high xylanase activity in the crude enzyme prepa-

rations from the pathogenic Fusarium strains grown in the

medium with rye cell wall is not surprising since in

graminaceous monocotyledonous plants, the primary and

secondary cell walls consist mainly of hemicellulose and

xylan (Belien et al. 2006; Carpita 1996; Hatsch et al. 2006;

Labavitch and Ray 1978).

There are reports suggesting that endoxylanases might

be pathogenicity factors for phytopathogenic microorgan-

isms. Indirect evidence for their role in pathogenesis has

been obtained by analyzing the production of cell wall-

degrading enzymes upon infection or by examining the

effect of purified endoxylanase on plant cells and infected

tissues (Belien et al. 2006; Brito et al. 2006; Polizeli et al.

2005). For instance, bacterial strains of Erwinia chrysant-

hemi that infect maize have been found to secrete more

endoxylanase activity than strains that infect only dicoty-

ledonous plants (Braun and Rodrigues 1993). Similarly,

fungal pathogens, such as Septoria nodorum, that infect

graminaceous monocotyledons have been reported to

secrete more xylan-degrading enzymes than pectinases

(Cooper et al. 1988; Lehtinen 1993). Fusarium-infected

barley has also been found to contain considerable amounts

of endoxylanase (Schwarz et al. 2002). Since xylans rep-

resent a large proportion of the hemicellulosic fraction of

cereal cell wall matrices, xylan-degrading enzymes are

expected to be important components of the offensive

arsenal of cereal pathogens (Cooper et al. 1988; Wanjiru

et al. 2002) and may have a role similar to that of pectic

enzymes in the infection of dicotyledons.

Arch Microbiol (2012) 194:653–665 659

123

Hydrolysis of PCW by crude enzymatic preparations

of Fusarium spp

Significant differences in the efficiency of PCW hydrolysis

were found both among the crude enzymatic preparations

obtained from 42-day autolyzing cultures of pathogenic

Fusarium isolates belonging to different species as well as

between the pathogenic and rhizospheric isolates of

Fusarium culmorum (Fig. 2).

The most efficient in this process, at pH 5.6, were the

enzymes released to the medium by the pathogenic isolate

of F. culmorum DEMFc37. The mixtures of hydrolases

contained in the growth media of F. graminearum DEM-

Fc36 and F. oxysporum DEMFc38 released from PCW,

under the same conditions, only 60 and 87%, respectively,

of the amount of reducing sugars released from this sub-

strate by the enzymes of Fusarium culmorum DEMFc37

(Fig. 2a).

The crude enzymatic preparation originating from the

culture of DEMFc37 was the most efficient in degrading

PCW in spite of the data shown in Table 1, indicating that

the activities of all the tested hydrolases in this preparation,

except for xylanases, were much lower than in the prepa-

ration obtained from the culture of F. oxysporum 38.

Although the hydrolytic efficiency of these enzymatic

preparations at pH 8.8 was lower than at pH 5.6, the dif-

ferences among the tested pathogenic isolates were the

same (Fig. 2b).

When the efficiency of hydrolysis of rye cell wall by

mixtures of enzymes released to medium by pathogenic

and rhizospheric F. culmorum strains was compared at pH

5.6 and 8.8, the enzymes of PGPF DEMFc2 released only

22 and 24%, respectively, of the amounts of reducing

sugars released by the preparations originating from

DEMFc37. The hydrolytic efficiency of the enzymes

originating from the DEMFc37 and DEMFc5 preparations

at both pH values tested was similar; however, at pH 8.8,

the activity of the DEMFc5 enzymes was significantly

higher. These findings strongly suggest that the earlier

reported (Jaroszuk-Sciseł et al. 2008, 2011) differences in

plant tissue colonization and induction of disease symp-

toms between the pathogenic strain DEMFc37 and the

rhizosphere isolates of Fusarium culmorum may be related

to differences in the efficiency of plant cell degradation by

the enzymes released by these strains to the PCW growth

medium.

The biggest variations in enzymatic activity of the

preparations obtained from the cultures of the tested

Fusarium spp. were found for xylanases (Table 1). These

enzymes are classified into two families, G10 and G11,

characterized by different molecular masses, pI, and

selectivity. Acidic high-molecular-mass endoxylanases

belong to family 10 of glycosyl hydrolases, and low-

molecular-mass basic endoxylanases belong to family 11.

Apart from their role in the degradation of xylan, family 11

fungal endo-b-1,4-xylanases are well-known proteinaceous

0

200

400

600

800

incubation time (hours)

μg o

f re

du

cin

g s

ug

ars

ml-1

a

0

200

400

600

800

0 24 48 72 96 0 24 48 72 96

incubation time (hours)

μg o

f re

du

cin

g s

ug

ars

ml-1

b

Fig. 2 Release of reducing sugars from plant cell wall (PCW) at

acidic (a) or alkaline (b) pH of the reacting mixture by crude

enzymatic preparations originating from 42-day cultures of Fusariumspp. (F. culmorum isolates DEMFc2 (filled circle), DEMFc5 (empty

circle), and DEMFc37 (filled triangle), F. graminearum isolate

DEMFc36 (empty square); and F. oxysporum isolate DEMFc38 (filledsquare)

660 Arch Microbiol (2012) 194:653–665

123

elicitors of defense response reactions in plants in a way

that is independent of their enzymatic activity (Enkerli

et al. 1999). The activity of alkaline xylanases in prepa-

rations obtained from Fusarium spp. cultures was usually

lower than that of acidic ones (Table 1).

The obtained results suggest that xylanase activity may

play a significant role in PCW degradation and pathogen-

esis. Experimental evidence demonstrating the requirement

for a single endoxylanase for virulence has been provided

by Brito et al. (2006), who showed that deletion of xyn11A

from Botrytis cinerea strongly affected its ability to infect

tomato leaves and grape berries. The appearance of sec-

ondary lesions on tomato leaves was delayed, and the

average size of those infections, that were actually

spreading, was reduced by more than 70%.

In the present study, the high positive value of the

Pearson correlation coefficient (R = ?0.962, p \ 0.05) and

the presence of a linear correlation (y = 1.96x - 351.8,

0

20

40

60

80

100

incubation time (hours)

μg o

f G

lcN

Ac

ml-1

a

0

20

40

60

80

100

0 24 48 72 960 24 48 72 96

0 24 48 72 96 0 24 48 72 96

incubation time (hours)

μg o

f G

lcN

Ac

ml-1

b

0

50

100

150

200

250

incubation time (hours)

μg o

f re

du

cin

g s

ug

ars

ml-1

c

0

50

100

150

200

250

incubation time (hours)

μg o

f re

du

cin

g s

ug

ars

ml-1

d

Fig. 3 Release of N-acetylglucosamine (GlcNAc) at acidic (a) or

alkaline (b) pH and reducing sugars at acidic (c) or (d) alkaline pH of

the reacting mixtures by crude enzymatic preparations originating

from cultures of F. culmorum isolates DEMFc2 (filled circle),

DEMFc5 (empty circle), and DEMFc37 (filled triangle), F. graminearum

isolate DEMFc36 (empty square), F. oxysporum isolate DEMFc38

(filled square), and from fungal cell walls (FCW) in a homologous

system (cell wall and enzymatic crude preparations originating from

the same isolate). Bars represent standard deviations of means of at

least three experiments

Arch Microbiol (2012) 194:653–665 661

123

R2 = 0.926, p \ 0.05) pointed to a close dependence

between the ability of the crude preparations originating

from the cultures of the tested Fusarium strains to

reduce the sugars released from PCW and their xylanase

activity.

Hydrolysis of FCW by Fusarium spp. crude enzymatic

preparations

The hydrolytic efficiency of enzymatic complexes was

tested in homologous (enzymes and cell wall substrates

0

20

40

60

80

100μg

of

Glc

NA

c m

l-1FCW of DEMFc2a

0

20

40

60

80

100FCW of DEMFc5

0

20

40

60

80

100FCW of DEMFc36

0

20

40

60

80

100

incubation time (hours)

incubation time (hours)

incubation time (hours)

incubation time (hours)

incubation time (hours)

incubation time (hours)

incubation time (hours)

incubation time (hours)

incubation time (hours)

incubation time (hours)

FCW of DEMFc38

0

20

40

60

80

100FCW of DEMFc37

0

50

100

150

200

250

mg

of

red

uci

ng

su

gar

s m

l-1

b

0

50

100

150

200

250

0

50

100

150

200

250

0

50

100

150

200

250

0 24 48 72 960 24 48 72 960 24 48 72 960 24 48 72 960 24 48 72 96

0 24 48 72 960 24 48 72 960 24 48 72 960 24 48 72 960 24 48 72 96

0

50

100

150

200

250

Fig. 4 Release of N-acetylglucosamine (GlcNAc) (a) and reducing

sugars (b) at acidic pH of the reacting mixtures by crude enzymatic

preparations originating from cultures of F. culmorum isolates

DEMFc2 (filled circle), DEMFc5 (empty circle), and DEMFc37

(filled triangle), F. graminearum isolate DEMFc36 (empty square),

F. oxysporum isolate DEMFc38 (filled square), and from fungal cell

walls (FCW) in a heterologous system (hydrolases and their substrates

coming from different isolates). Bars represent standard deviations of

means of at least three experiments

662 Arch Microbiol (2012) 194:653–665

123

originating from the same isolate) (Fig. 3a, b) and heter-

ologous (Fig. 4a, b) (hydrolases and their substrates orig-

inating from different isolates) systems.

The activities of hydrolases contained in crude prepa-

rations obtained from the supernatants of fungal cultures

grown in media with their own cell walls as C sources are

shown in Table 1b.

In homologous systems, F. oxysporum DEMFc38 was

found to be the most efficient of the tested pathogenic

strains at both pH values (5.6 and 8.8). The efficiencies of

release of reducing sugars in the homologous system of

F. graminearum DEMFc36 at pH 5.6 and 8.8 were 91 and

95%, respectively, of those of F. oxysporum (Fig. 3b).

However, there was a much larger difference in the

amounts of GlcNAc released in these two homologous

systems (30.6% at pH 5.6 and 24% at pH 8.8) (Fig. 3a).

The efficiency of release of reducing sugars in the

homologous system of pathogenic F. culmorum DEMc37 at

both pH values was the lowest among the tested pathogens.

Among the homologous systems, the rhizosphere isolate

of F. culmorum DEMFc2 (PGPF) was the most efficient in

the hydrolysis of FCW at both pH values tested. The

amount of reducing sugars released at pH 5.6 accounted for

177% of the amount released in the most efficient homol-

ogous system of the pathogenic F. oxysporum isolate.

When the amounts of reducing sugars released from the

same substrates by hydrolases of the various individual

pathogens (heterologous system) were compared, no sig-

nificant differences were found between the efficiencies of

the enzymes originating from the cultures of DEMFc36

and DEMFc38. Much less efficient in this process were the

enzymes present in the supernatant of the autolyzed culture

of the isolate DEMFc37 (Fig. 3b).

When the efficiency of Fusarium cell wall hydrolysis in

homologous (Fig. 3a, b) and heterologous systems

(Fig. 4a, b) was compared, no significant differences were

found in the amounts of reducing sugars and GlcNAc

released in the homologous system of the rhizosphere

isolate DEMFc2 and the heterologous systems consisting

of hydrolases of DEMFc2 and cell walls of the pathogenic

strains F. culmorum DEM Fc37 or F. graminearum

DEMFc36 or F. oxysporum DEMFc38 at pH 5.6 and 8.8.

The results presented here indicate that the efficiency of

FCW degradation by the individual strains is mainly rela-

ted to the activities of two hydrolases released by them to

the medium: glucanases and chitinases. In general, glu-

canase activities in the crude preparations used to hydro-

lyze FCW were about 10 times higher than chitinase

activities, and the amounts of reducing sugars released by

glucanases from FCW were higher than the amounts of

GlcNAc (Table 1; Figs. 3a, b, 4a, b). There are reports

showing that effective degradation of FCW is catalyzed by

the synergistic action of chitinases releasing GlcNAc and

b-1,3-glucanases releasing reducing sugars (Adams 2004;

Alfonso et al. 1992, 1995a, b; Isaak and Gokhale 1982;

Skujins et al. 1965; Lahoz et al. 1976; Santamaria et al.

1995). The highest activities of chitinases and b-1,3-glu-

canases were found in the preparation from the culture of

the PGPF strain DEMFc2. This strain also released the

highest amounts of GlcNAc and reducing sugars from

FCW (Table 1; Figs. 3a, b, 4a, b). By contrast, Lynch et al.

(1985) found that culture filtrates of F. tricinctum having

maximum b-glucanase activity but no chitinase activity

were efficient in liberating protoplasts from the young

mycelium of the same species and in lysing F. oxysporum

mycelium. A study by Perez-Leblic et al. (1982) also

indicated that b-1,3-glucanase was the only enzyme present

at high levels in all of the extensively autolyzed cultures

(Neurospora crassa, Botrytis cinerea, Polystictus versi-

color, Aspergillus nidulans, Schizophyllum commune,

Aspergillus niger and Mucor mucedo) those authors tested.

The highest effectiveness of DEMFc2 in degrading

FCW can explain the protective effect of pre-inoculation of

rye with this isolate against infection by the pathogenic

strain DEMFc37 (Jaroszuk-Sciseł et al. 2008). This is

because one product of chitin degradation by the PGPF

isolate is an oligosaccharide with elicitor activity which

could induce plant resistance (Shibuya and Minami 2001).

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%DEMFc2 DEMFc5 DEMFc37

% o

f F

CW

dry

wei

gh

t

GCH G1 G2 GGM

Fig. 5 Cell wall fractions of the particular Fusarium culmorumisolates: (GCH) – b-(1,3)-glucan–chitin complex fraction—F4;

(G1) – fraction of a/b-(1,3)-glucan weakly associated with chitin—

F1I; (G2) – fraction of a/b-(1,3)-glucan strongly associated with

chitin—F3; (GGM) – b-(1,5)-galacto-manno-glucan fraction—F1S

Arch Microbiol (2012) 194:653–665 663

123

When the efficiency of FCW hydrolysis in homologous

and heterologous systems was compared, the obtained

results (Figs. 3a, b, 4a, b) suggested that the degree of

FCW hydrolysis was affected by the composition and

activities of enzymatic compounds present in the reacting

mixture rather than the origin of the FCW used. Santamaria

et al. (1995) also found that the level of efficiency of

F. oxysporum sp. lycopersici cell wall lysis was determined

by the origin of the hydrolases used.

The highest positive Pearson correlation coefficient value

(R = ?0.977) and a linear correlation (y = 2.87x - 43.584,

R2 = 0.955, p \ 0.05) was found between chitinase

activity in enzymatic preparations obtained from culture

supernatants of the Fusarium spp. strains and the efficiency

of FCW hydrolysis in homologous and heterologous

systems.

Chemical fractionation of the tested Fusarium spp. cell

walls indicated that there were no significant differences in

the contents of glucochitin (GCH), glucanases weakly

(F1I) and strongly (F3) associated with chitin, and galacto-

manno-glucan (F1S) fractions between the tested rhizo-

sphere and pathogenic Fusarium culmorum strains (Fig. 5).

The chemical composition of the fungal cell wall is species

specific but also varies depending on the development and

morphological stage as a result of aging (Bowman and Free

2006; Ruiz-Herrera 1991).

Acknowledgments The scientific research was financed from sci-

ence funding resources as a personal research project no. N N310

441338 in the years 2010–2013.

References

Adams DJ (2004) Fungal cell wall chitinases and glucanases.

Microbiology 150:2029–2035

Akimitsu K, Isshiki A, Ohtani K, Yamamoto H, Eshel D, Prusky D

(2004) Sugars and pH: a clue to the regulation of fungal cell

wall-degrading enzymes in plants. Physiol Mol Plant Pathol

65:271–275

Alef K, Nannipieri P (1995) Methods in applied soil microbiology and

biochemistry. Academic Press, New York, USA, pp 360–361

Alfonso C, del Amo F, Nuero OM, Reyes F (1992) Physiological and

biochemical studies on Fusarium oxysporum f. sp. lycopersicirace 2 for its biocontrol by nonpathogenic fungi. FEMS

Microbiol Lett 99:169–174

Alfonso C, Nuero OM, Santamaria F, Reyes F (1995a) Purification of

a heat-stable chitin deacetylase from Aspergillus nidulans and its

role in cell wall degradation. Curr Microb 30:49–55

Alfonso C, Santamaria F, Nuero OM, Prleto A, Leal JA, Reyes F

(1995b) Biochemical studies on the cell wall degradation of

Fusarium oxysporum f. sp. Iycopersici race 2 by its own lytic

enzymes for its biocontrol. Lett Appl Microbiol 20:105–109

Annis SL, Goodwin PH (1997) Recent advances in the molecular

genetics of plant cell wall-degrading enzymes produced by plant

pathogenic fungi. Eur J Plant Pathol 103:1–14

Apel PC, Panaccione DG, Holden FR, Walton JD (1993) Cloning and

targeted gene disruption of XYL1, a beta 1,4-xylanase gene from

the maize pathogen Cochliobolus carbonum. Mol Plant Microbe

Interact 6:467–473

Armitage P, Berry G (1987) Statistical methods in medical research.

Blackwell Science Publications, Oxford

Belien T, Van Campenhout S, Robben J, Volckaert G (2006)

Microbial endoxylanases: effective weapons to breach the plant

cell wall barrier or, rather, triggers of plant defense systems?

Mol Plant Microbe Interact 19:1072–1081

Boothby D, Magreola NO (1984) Production of polysaccharide

degrading enzymes by Cochliobolus sativus and Fusariumculmorum grown in liquid culture. Trans Br Mycol Soc 83:275–

280

Bowman SM, Free SJ (2006) The structure and synthesis of the fungal

cell wall. BioEssays 28:799–808

Braun EJ, Rodrigues CA (1993) Purification and properties of an

endoxylanase from a corn stalk rot strain of Erwinia chrysant-hemi. Phytopathology 83:332–338

Brito N, Espino JJ, Gonzalez C (2006) The endo-beta-1,4-xylanase

xyn11A is required for virulence in Botrytis cinerea. Mol Plant

Microbe Interact 19:25–32

Carpita NC (1996) Structure and biogenesis of the cell walls of

grasses. Annu Rev Plant Physiol Plant Mol Biol 4:445–476

Carpita NC, Gibeaut DM (1993) Structural models of primary cell

walls in flowering plant: consistency of molecular structure with

the physical properties of the walls during growth. Plant J 3:1–30

Cohen CK, Fox TC, Garvin DF, Kochian L (1998) The role of iron-

deficiency stress responses in stimulating heavy-metal transport

in plants. Plant Physiol 116:1063–1072

Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families

and extremophilic xylanases. FEMS Microbiol Rev 29:3–23

Cooper RM, Longman D, Campbell A, Henry M, Lees PE (1988)

Enzymic adaptation of cereal pathogens to the monocotyledon-

ous primary wall. Physiol Mol Plant Pathol 32:33–47

Degefu Y, Fagerstrom R, Kalkkinen N (1995) Purification and partial

characterization of xylanase from fungal pathogen Helmintho-sporium furcicum (Pass). Eur J Plant Pathol 101:291–299

Degefu Y, Paulin L, Lubeck PS (2001) Cloning, sequencing and

expression of a xylanase gene from the maize pathogen

Helminthosporium turcicum. Eur J Plant Pathol 107:457–465

Douaiher M-N, Nowak E, Dumortier V, Durand R, Reignault PH,

Halama P (2007) Mycosphaerella graminicola produces a range

of cell wall-degrading enzyme activities in vitro that vary with

the carbon source. Eur J Plant Pathol 117:71–79

Ebel J, Casio EG (1994) Elicitors of plant defense responses. Int Rev

Cytol 148:1–36

Enkerli J, Felix G, Boller T (1999) The enzyme activity of fungal

xylanase is not necessary for its elicitor activity. Plant Physiol

121:391–397

Furman-Matarasso N, Cohen E, Du Q, Chejanovsky N, Hanania U,

Avni A (1999) A point mutation in the ethylene-inducing

xylanase elicitor inhibits the b-1-4-endoxylanase activity but not

the elicitation activity. Plant Physiol 121:345–351

Giesbert S, Lepping H-B, Tenberge KB, Tudzynski P (1998) The

xylanolytic system of Claviceps purpurea: cytological evidence

for secretion of xylanases in infected rye tissue and molecular

characterization of two xylanase genes. Phytopathology 88(10):

1020–1030

Gomez-Miranda B, Guettero C, Leal JA (1984) Effect of culture age

an cell wall polysaccharides of Penicillium allahabadence. Exp

Mycol 8:298–303

Gomez-Miranda B, Prieto A, Leal JA (1990) Chemical composition

and characterization of a galactomannoglucan from Gliocladiumviride. FEMS Microbiol Lett 70:331–336

Hatsch D, Phalip V, Petkovski E, Jeltsch J-M (2006) Fusariumgraminearum on plant cell wall: no fewer than 30 xylanase genes

transcribed. Biochem Biophys Res Commun 345:959–966

664 Arch Microbiol (2012) 194:653–665

123

Hope CFA, Burns RG (1987) Activity, origins and location of

cellulase in silt loam soil. Biol Fert Soils 5:164–170

Isaak S, Gokhale AV (1982) Autolysis: a tool for protoplast

production from Aspergillus nidulans. Trans Br Mycol Soc 78:

389–394

Jaroszuk-Sciseł J, Kurek E, Winiarczyk K, Baturo A, Łukanowski A

(2008) Colonization of root tissues and protection against

fusarium wilt of rye (Secale cereale) by nonpathogenic rhizo-

sphere strains of Fusarium culmorum. Biol Control 45:297–307

Jaroszuk-Sciseł J, Kurek E, Rodzik B, Winiarczyk K (2009)

Interactions between rye (Secale cereale) root border cells

(RBCs) and pathogenic and nonpathogenic rhizosphere strains of

Fusarium culmorum. Mycol Res 113:1053–1061

Jaroszuk-Sciseł J, Kurek E, Słomka A, Janczarek M, Rodzik B (2011)

Activities of cell wall degrading enzymes in autolyzing cultures

of three Fusarium culmorum isolates: growth promoting, dele-

terious and pathogenic to rye (Secale cereale). Mycologia

103(5):929–945

Kang Z, Buchenauer H (2000) Ultrastructural and cytochemical

studies on cellulose, xylan and pectin degradation in wheat

spikes infected by Fusarium culmorum. J Phytopathol 148:

263–275

Keon JPR, Byrde RJW, Cooper RM (1987) Some aspects of fungal

enzymes that degrade plant cell walls. In: Pegg GF, Ayres PG

(eds) Fungal infection of plants. University Press, Cambridge,

pp 133–157

Kurek E, Jaroszuk J (1997) Changes in the number of Fusariumpropagules introduced to soil. Polish J Soil Sci 30:63–69

Labavitch TM, Ray PM (1978) Structure of hemicellulosic polysac-

charides of Avena sativa coleoptile cell walls. Phytochemistry

17:933–937

Lahoz R, Reyes F, Beltra R, Garcia-Tapia C (1976) Lytic enzymes in

the autolysis of filamentous fungi. Mycopathologia 60:45–49

Lalaoui F, Halama P, Dumortier V, Paul B (2000) Cell wall-

degrading enzymes produced in vitro by isolates of Phaeosp-haeria nodorum differing in aggressiveness. Plant Pathol

49:727–733

Leal JA, Gomez-Miranda B, Prieto A, Bernabe M (1992) Chemical

and structural similarities in wall polysaccharides of some

Penicillium, Eupenicillium and Aspergillus. FEMS Microbiol

Lett 90:165–168

Lehtinen U (1993) Plant cell wall degrading enzymes of Septorianodorum. Physiol Mol Plant Pathol 43:121–134

Lynch PT, Collin HA, Isaac S (1985) Use of autolytic enzyme for

isolation of protoplasts from Fusarium tricinctum hyphae. Trans

Br Mycol Soc 84:473–478

Martin JP (1950) Use of acid rose Bengal and streptomycin in the

plate methods for estimating soil fungi. Soil Sci 38:215–220

Montesano M, Brader G, Palva ET (2003) Pathogen derived elicitors:

searching for receptors in plants. Mol Plant Pathol 4:73–79

Nelson N (1944) A photometric adaptation of the Somogyi method

for the determination of glucose. J Biol Chem 153:375–380

Perez-Leblic MI, Reyes F, Martinez MJ, Lahoz R (1982) Cell wall

degradation in the autolysis of filamentous fungi. Mycopatho-

logia 80:147–155

Phalip V, Goubet F, Carapito R, Jeltsch J-M (2009) Plant cell wall

degradation with a powerful Fusarium graminearum enzymatic

arsenal. J Microbiol Biotechnol 19:573–581

Polizeli MLTM, Rizzatti ACS, Monti R, Terenzi HF, Jorge JA,

Amorim DS (2005) Xylanases from fungi: properties and

industrial applications. Appl Microbiol Biotechnol 67:577–591

Reyes F, Byrde RJW (1973) Partial purification and properties of a

p-N-acetylglucosaminidase from the fungus Sclerotinia fructi-gena. Biochem J 131:381–388

Reyes F, Lahoz R, Cornago P (1977) Autolysis of Neurospora crassain different culture conditions and release of b-N-acetylgluco-

saminidase and chitinase. Trans Br Mycol Soc 68:357–361

Rodriguez-Kabana R, Godoy G, Morgan-Jones G, Shelby RA (1983)

The determination of soil chitinase activity; conditions for assay

and ecological studies. Plant Soil 75:95–106

Rossner H (1991) Bestimmung der Chitinase-Aktivitat. In: Schinner

F, Ohlinger R, Kandeler E (eds) Bodenbiologische Arbeitsme-

thoden. Springer, Berlin, pp 66–70

Ruiz-Herrera J (1991) Biosynthesis of b-glucans in fungi. Antonie

Van Leeuwenhoek 60(2):73–81

Santamaria F, Nuero OM, Alfonso C, Prieto A, Leal JA, Reyes F

(1995) Cell wall degradation of Fusarium oxysporum f. sp.

lycopersici race 2 by lytic enzymes from different Fusariumspecies for its biocontrol. Lett Apll Microbiol 20:385–390

Schwarz PB, Jones BL, Steffenson BJ (2002) Enzymes associated

with Fusarium infection of barley. J Am Soc Brew Chem

60(3):130–134

Shibuya N, Minami E (2001) Oligosaccharide signalling for defence

responses in plant. Physiol Mol Plant Pathol 59:223–233

Skujins JJ, Potgieter HJ, Alexander M (1965) Dissolution of fungal

cell walls by a streptomycete chitinase and b-(1–3) glucanase.

Arch Biochem Biophys 111:358–364

Somogyi M (1945) A new reagent for determination of sugars. J Biol

Chem 160:61–68

Wanjiru WM, Zhensheng K, Buchenauer H (2002) Importance of cell

wall degrading enzymes produced by Fusarium graminearumduring infection of wheat heads. Eur J Plant Pathol 108:803–810

Wolska-Mitaszko B, Jaroszuk-Sciseł J, Pszeniczna K (2007) Isoforms

of trehalase and invertase of Fusarium oxysporum. Mycol Res

111:456–465

Wu SC, Kauffmann S, Darvill AG, Albersheim P (1995) Purification,

cloning and characterization of two xylanases from Magnapor-the grisea, the rice blast fungus. Mol Plant-Microbe Interact

8:506–514

Wu SC, Halley JE, Luttig C, Fernekes LM, Gutierrez-Sanchez G,

Darvill AG, Albersheim P (2006) Identification of an endobeta-

1,4-D-xylanase from Magnaporthe grisea by knockout analysis,

purification, and heterologous expression. Appl Environ Micro-

biol 72:986–993

Arch Microbiol (2012) 194:653–665 665

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