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Chitinolytic activity at low temperature of an Antarctic strain (A3) of Verticillium lecanii
O INSTrrUT PASTEUR/ELSEVIER Paris 1998
Res. Microbiol. 1998, 149, 289-300
Chitino|ytic activity at low temperature of an Antarctic strain (A3) of Verticillium lecanii M. Fenice, L. Selbmann, R. Di Giambattista and F. Federici ~*) Dipartimento di Agrobiologia e Agrochimica, University of Tuscia, Via S. Camillo de Lellis, 1-01100, Viterbo (Italy)
SUMMARY The chitinolytic activity of Verticillium cfr. lecanfi A3, a strain isolated from continental Antarctica, was compared to those of two selected strains of Trichoderma harzianum. After 72 h of incubation at 25 oC in media containing chitin as the sole carbon source, all strains showed the same enzyme activity (ca. 230 mU/ml); at 15°C, the levels of enzyme activity of the three strains were similar to those obtained at 250C. At 5oc, in contrast, the activity of V. lecanfi was ca. 4 times higher than those of both strains of T. harzianum (203 and 57 mU/ml, respectively; incubation time 144 h}. The chitinase of V. lecanii, purified by preparative isoelectric focusing and ion-exchange chromatography, was shown to be a glycoprotein with apparent molecular weight of 45 kDa and isoelectric point of 4.9. The enzyme was active over a broad range of temperatures (5-60"C): at 5"C, its relative activity was still 50% of that recorded at 40°C (optimal temperature). V. lecanii and its purified chitinase showed clear inhibitory effects on the growth of some test moulds such as Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor and Penicillium verrucosum: observations under the light and scanning electron microscopes revealed that growth inhibition was accompanied by myceliai damage and cell lysis.
Key-words: Chitin, Verticillium lecanii, Trichoderma harzianum; Low temperature, Chitinolytic activity, Antifungal action.
INTRODUCTION Due to undesirable side effects on the environment and human health, the use of chemical pesticides and food preservatives has been widely criticized in recent years (Chet et al., 1993; Lorito et al., 1994b). As a consequence, microorganisms have been studied in order to develop safer alternatives to chemical treatments (Chet et
Submitted November 13, 1997, accepted February 5, 1998. (*) Corresponding attthor.
al., 1993). Among fungi, Trichoderma harzianum, T. (Gliocladium) virens, Penicillium sp., A c r e m o n i u m alternatum and A p h a n o c l a d i u m album have been particularly investigated (Studer et al., 1992; Malathrakis and Kritsotaki, 1992; Malathrakis and Klironomou, 1992; Chet et al., 1993 ; Di Pietro et aL, 1993 ; Lorito et al., 1993, 1994a,b ; Schirmbock et ai., 1994). The large majority of these studies, however, have con-
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M. FENICE ET AL.
cerned T. harzianum and Z virens, mycoparasites that produce chitinolytic enzymes involved in host cell degradation (Chet et al., 1993; Haran et aL, 1994; Lorito et al., 1994a,b; Schirmbock et al., 1994). Similar evidence is lacking for other fungal organisms. This paper deals with the chitinolytic activity at different growth temperatures of Verticillium lecanii strain A3 isolated from continental Antarctica (Fenice et al., 1996) as compared to the activity of two selected strains of T. harzianum, known for their chitinolytic activity (Harman et ai, 1989; Tronsmo, 1989). The isolation site of this fungus is an extreme terrestrial environment which can be colonized only by organisms with a high level of adaptation (Fenice et al., 1997). Thus, the characteristics of such an organism may be of interest for specific biotechnology fields, i,e. treatment of chitin-rich wastes at low temperature and/or biocontrol of phytopathogens in cold environments. In this context, the antifungal activities of V. lecanii and its purified chitinase against some spoiling agents of refrigerated foods, such as Mucor plumbeus, Penicillium verrucosum, Aspergillus versicolor and Cladosporium cladosporioides have also been investigated.
(Detroit, MI, USA). All other chemicals were of analytical grade. Microrganisms V. cfr. lecanii A3, isolated from moss samples collected in continental Antarctica, was obtained from the Dipartimento di Scienze Ambientali, University of Tuscia, Viterbo, Italy. T. harzianum P1 and T. harzianum T22 were obtained from the lstituto di Patologia Vegetale, University of Napoli "Federico 11", Portici, Naples, Italy. M. plumbeus, P. verrucosum, A. versicolor and C. cladosporioides were from the culture collection of the Life Science Department, Nestl~ Research Center (Lausanne, Switzerland). All strains are stocked in the culture collection of the Dipartimento di Agrobiologia e Agrochimica, University of Tuscia, Viterbo, Italy. During the study, the cultures were maintained on MEA at 4-6°C and subcultured every month. Chitin preparation
Colloidal chitin was prepared by treating chitin from crab shells as reported by Hankin and Anagnostakis (1975). After swelling, chitin was resuspended in water and sterilized by autoclaving (121°C, 20 min). The solid content of the sterile chitin suspension was evaluated by dry weight determinations. Glycol chitin was prepared by reacetylation of glycol chitosan using the method of Molano et al. (1977).
MATERIALS AND METHODS Culture media and growth conditions Chemicals
Chitin (from crab shells), p-nitrophenyl-B-D-Nacetylglucosaminide, p-nitrophenyl-B-D-N-N'-diacetylchitobiose, p-nitrophenyl-B-D-N-N'-N"-triacetylchitotriose, glycol chitosan and xylan were from Sigma Chemical Co. (St. Louis, MO, USA). Chitosan (low, medium and high molecular weight), cellulose acetate and CM-cellulose were from Fluka BioChemika (Buchs, CH). Malt extract agar (MEA) and potato dextrose agar (PDA) were from Oxoid (Unipath Ltd., Basingstoke, Hampshire, UK), yeast nitrogen base (YNB) was from Difco Laboratories
BM cfr. EDTA IEF MEA
= = = = =
basal medium. confrontation. ethylenediamine tetraacetate. isoelectric focusing. malt extract agar.
The basal culture medium (BM) was as follows (w/v): YNB, 1% and colloidal chitin, 1%, pH 5.5. The solid medium for dual cultures was PDA, 4 %. The liquid medium for microcultures was YNB, 1% and glucose, 1% (YG). All media were sterilized by autoclaving (121°C, 20 min). Shaken cultures
After inoculation (final concentration ca. 5.0 x 105 conidia/ml), cultures were incubated on a rotary shaker, 200 cycles/min, tor 9 days at 5, 15 and 25°C.
PDA SDS-PAGE
= =
SEM YNB
= =
potato dextrose agar. sodium dodecyl sulphatc/ptdyacrylamide gel elcctrophoresis. scanning electron microscope. yeast nitn~gen base.
CHITINOLYTIC ACTIVITY OF VERTICILLIUM LECANII A3 Samples were taken every 12-24 h and, alter centrifugafion (10,000 g, 10 min), the supernatants were used for the enzyme assay.
Dual cultures Petri dishes (10-cm diameter) filled with 20 ml of PDA were inoculated (punctiform inocula, 4 cm apart) with V. iecanii and each of the test organisms, and incubated at 25 and 5°C. In order to investigate fungal interactions and the occurrence of mycoparasitism, the mycelial structures in the contact zone of the two colonies were observed under light and scanning electron microscope (SEM).
Microcultures Thirteen l.tl of conidiospore suspensions (5 x 10 3 conidia/ml) in YG were pipetted onto sterile microscope slides and added with 13 lal of a filter sterilized solution ef purified chitinase (1.0, 2.0 and 5.0 U/ml); slides were then placed in sterile petri dishes and incubated at 25°C for 24 h in a wet thermostated room. After 15 and 24 h of incubation, the microcultures were observed under the light microscope. For the controls, sterile distilled water was used in place of the enzyme solutions.
Enzyme recovery and purification All procedures were carried out at 4°C.
Step 1: ammonium sulphate fractionated precipitation The crude enzyme solution was centrifuged (5,000 g, 10 min) and filtered (GFC membrane Whatman, UK) to remove the mycelium. The filtrate was brought to 30% saturation with ammonium sulphate, left for 2 h at 4°C and then centrifuged (5,000 g, 10 min). The supernatant was then brought to 75 % saturation with the same salt and left overnight at 4°C. The precipitate formed was collected by centrifugation (8,000 g, 15 min), dissolved in 15 ml of 5 mM citrate-phosphate buffer pH 5.0 and dialysed overnight against several volumes of the same buffer. The final enzyme solution, which contained ca. 90% of the initial chitinolytic activity, was concentrated by lyophilization. The lyophilized material had an activity of 2.3 U/mg of protein.
Step 2: preparative isoelectric fi~cusing (Rotofor) After resuspension in distilled water, the ]y~ philized enzyme from step 1 was applied t~ a
291
"Rotofor" apparatus (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's directions, and R o t o l y t e s ( B i o - R a d Laboratories) (range 4.5-6.1) at a gradient of 4.5-5.7 were used. Peak fractions were collected, pooled and concentrated by centrifugation (6,000 g, 10 min) with a "Centricon 10000" membrane system (Amicon, USA).
Step 3: ion-exchange column chromatography The dialysed enzyme from step 2 was directly applied to an "Econo-pack Q" cartridge (Q-sepharose) (Bio-Rad Laboratories) equilibrated with 20 mM tris-HCl buffer pH 8.0 and eluted with a 3step gradient of the same buffer containing 0-1 M NaCI at a flow rate of 1 ml/min. Fractions (2.0 ml each) containing the chitinolytic activity were pooled and concentrated as above.
Enzyme characterization The effect of pH on the enzyme activity was determined by varying the pH of reaction mixtures using 50 mM glycine/HCl buffer (pH 1.0-3.0), 50 mM citrate phosphate buffer (pH 3.5-7.0) and 50 mM phosphate buffer (pH 7.0-8.0). The denaturing effects of pH were investigated by incubating the enzyme solution for 1 h at 25°C in the range of pH values from 3.0 to 6.0. The effect of temperature on the enzyme activity was determined at pH 4.0 in the range of temperatures from 5 to 80°C. Temperature stability was evaluated by incubating the enzyme solution at 40°C and pH 4.0 for 10 h: samples were taken every hour and the residual enzyme activity determined. The effects of metal ions such as K +, Ca ++, Fe ++, Mg ++, Mn +÷, Zn ++, sodium azide and EDTA on the apparent enzyme activity were determined after preincubation (37°C, 30 min) of the enzyme solution with 10 and 100 mM of the above compounds.
Electrophoresis Sodium dodecyl sulphate/polyacry]amide gel electrophoresis (SDS-PAGE) was used to check the purity of protein and to determine the molecular mass of the purified enzyme under denaturing conditions using a 12.0% polyacrylamide gel, according to Laemmli (1970). After electrophoresis, the gels were stained with Coomassie brilliant blue R-250 (Pharmacia, Sweden) or by the periodic acid Schiff reagent for glycoproteins (Zacharius eta/., 1969). Molecular markers (low range) were from Bio-Rad Laboratories (CA).
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M. F E N I C E E T AL.
Isoelectric focusing (IEF) The isoelectric point (pl) of the purified chitinase was determined using a "Multiphor II" cell (Pharmacia, Sweden) and a precasted polyacrylamide gel "Pagplate" (Pharmacia, Sweden) with ampholine pH range 4.0-6.5. Broad-range (3.5-10) IEF gel protein calibration kits were from Pharmacia (Sweden); stainings for protein were as described for SDSPAGE,
V. lecanii reached the same level of chitinolytic activity (228 mU/ml) at the same incubation time (72 h); after this time, however, its enzyme activity began to decrease. The maximal activity of T. h a r z i a n u m P I was c o n s i d e r a b l y lower (121 mU;ml) and was reached only after 120 h of incubation (fig. IA). At 15°C, the three strains showed the same levels of activity as above, but production was delayed ca. 48 h (fig. IB). At 5°C, V. lecanii reached a level of enzyme activity
Protein estimation Protein concentration was estimated spectrophotometrically at 595 nm using bovine serum albumin as a standard protein, by the Bio-Rad Laboratories (CA) procedure (Bradford, 1976).
25O
A
200
Enzyme assay
150
Chitinolytic activity was determined as previously reported (Fenice et al., 1996) by measuring the amount of reducing sugars liberated from colloidal chitin by the enzyme activity; N-acetyl-Dglucosamine was used for the standard curve. Under the assay conditions, one unit (U) of enzyme activity was defined as the amount of enzyme which released 1 lamol of N-D-acetylglucosamine per milliliter per minute.
!00
50 0
|
|
I
i
250 2(X) 150
Scanning electron microscopy Samples from different places of petri dishes containing dual cultures were cut with a sharp lancet and treated as reported by Onofri et al. (1980). A "Jeol 5002 JSM" (Jeol, Japan) SEM was used.
al
5o
0
250
RESULTS
Chitinolytic act.~vity of V. lecanii at different growth temperatures V. lecanii was grown at different temperatures (5, 15 and 25°C) and its chitinolytic activity compared to those of two selected strains of T. harzianum (T. harzianum PI and Z harzianum T22); results are reported in figure 1.
At 25°C, T. harzianum T22 reached its maximal activity (230 mU/ml) after 72 h of incubation, and the activity remained almost stable for 72 more hours to decrease markedly thereafter.
I
.....
1
I
c
200
150
100
50
v
24
48
72
9b
120
144
168
192
216
Time (hours)
Fig. 1. Chitinolytic activity of V. lecanii (0), T. harziamlm PI (V) and T. harzianmn T22 (O) grown at 25 (A), 15 (B) and 5°C (C). Values represent the average of three experiments and error bars indicate standard deviations.
CHITINOLYTIC ACTIVITY OF VERTICILLIUM LECANI] A3
(203 mU/ml after 144 h of incubation) very close to that obtained at 25°C. T. harzianum T22 and T. harzianum PI, on the contrary, showed much lower levels of activity (22 and 57 mU/rnl, respectively), and the same fermentation time (fig. 1C).
Enzyme purification and charactedzation The crude enzyme from V. lecanff culture was fractionated by preparative IEF (Rotofor) and ion-exchange chromatography (Q-sepharose). After Rotofor separation, the chitinolytic activity was found in fractions with pH about 5.0. These fractions were combined and applied to ion exchange, eluted within the gradient (ca. 20% of 1M NaCI) and recovered as a single peak. The recovery yield was 14% of the initial enzyme activity (table I). After ion-exchange chromatography, fractions showing chitinolytic activity were pooled and submitted to SDS-PAGE: a single band was obtained, with apparent molecular weight of 45 kDa (fig. 2). After staining with the periodic acid Schiff reagent (Zacharius et al., 1969), the chitinase appeared to be a glycoprotein having an isoelectric point of 4.9 (data not shown). The optimal pH for activity was 4.0; at pH 3.5 and 4.5 the enzyme retained about 53 and 66 % of the activity shown at pH 4.0, respectively. The denaturing effect of pH was tested by incubating the enzyme solution for lh at 25°C in the pH range 3.0-6.0: the enzyme was most stable at pH 5.0. The purified chitinase was active over the temperature range 5-60°C with a maximum at 40°C; at 5°C, the enzyme retained 50% of the activity shown at 40°C. After 1, 4 and 8 h of incubation at optimal pH and temperature, the
293
enzyme maintained 80, 50 and 5 % of the initial activity, respectively. The effects of different metal ions, EDTA and NaN 3 on the enzyme activity are reported in table lI. At 10 mM, K +, Fe ++, NaN 3 and EDTA did not produce any inhibitory effects, while the other substances caused 30 % inhibition. At 100 mM, the inhibitory effects were more evident: Zn ÷+, ML ~+ and EDTA caused an activity reduction of about 50 % and with Mg ++ the residual activity was about 30%. The chitinolytic enzyme of V lecanii hydroiysed colloidal a~,'7 glycol chitin more easily than other chitins (table III). There was no activity on either the celluloses or NN'-diacetylchitobiose; xylan and chitosans were degraded to a minor extent.
Ant~ungal activity of V. lecanii and its purified chitinase
V. lecanii and its purified chitinase showed clear inhibitory effects on a series of test moulds (M. plumbeus, C. cladosporioides, P verrucosum and A. versicolor) (table IV and figs. 3-6).
Figure 3A shows the appearance of a dual culture of V. lecanii and M. plumbeus after 15 days of incubation at 25°C. An area of lysis of the mycelium of M. plumbeus was present in the contact zone between the two microorganisms (centre of the plate) (fig. 3A). Also, V. lecanii (white aerial mycelium) almost completely overgrew M. plumbeus (light grey mycelium with black spots of sporangia shown in figure 3B). Furthermore, some structures, such as the sporangiophores, appeared to be damaged, with a considerable reduction in size (fig. 3C).
Table I. Purification of the chitinolytic activity of V. lecanii.
Step
Protein (rag)
Chitinase (U)
Sp. activity (U/mg)
Yield (%)
Purification (fold)
(NH4)2SO4 Rotofor Q-sepharose
68.3 9.18 0.043
204.4 89.1 28.0
2.99 9.70 65! .2
100 44 14
1 3 218
294
M. FENICE ET AL.
97A
66,2
Q 31
2t,S
t
2
$
KDa
Fig. 2. SDS-PAGE (12%) electrophoresis of the purified chitinolytic enzyme of V. lecanii. Lane 1= ion exchange (Q-sepharose); lane 2 = Rotofor; lane 3=ammonium sulphate precipitate. Molecular weight markers were bovine albumin (66 kDa), egg albumin (45 kDa), carbonic anhydrase (29 kDa) and trypsin inhibitor (20 kDa). Gel was stained with Coomassie blue.
Figure 4 shows a dual culture of V. lecanii and C. cladosporioides: the latter was almost overwhelmed by the mycelial growth of the former. Similar results were obtained with dual cultures of V. lecanii and P. verrucosum or A. versicolor (pictures not shown).
Samples were collected from the interaction region of dual cultures of V. lecanii and M. p l u m b e u s and o b s e r v e d u n d e r the SEM (fig. 5). V. lecanii established a very close contact with M. plumbeus: it coiled around the host mycelia (fig. 5-1) and penetrated into its hyphae (figs. 5-2 and 5-3), sporangia and sporangiospores (figs. 5-4 and 5-8). The mycoparasitic action of V. lecanii on M. plumbeus produced hyphal loss of turgor and degradation (figs. 5-5, 5-6 and 5-7) and spore deflating (fig. 5-8). The same results were obtained when dual cultures were incubated at 5°C; at this temperature, however, 30 days of incubation were necessary to observe the mycoparasitic effect (data not shown). Microcultures of the above test organisms, treated with 2 U/ml of the purified chitinase from V. lecanii, showed inhibition of spore germination, mycelium vacuolization and lysis, cell wall disruption, protoplast formation and bursting. M. p l u m b e u s , after 15 h of i n c u b a t i o n , showed r e d u c e d or no spore g e r m i n a t i o n . After 24 h, an inhibitory effect began to be evident that consisted in m a r k e d cell wall damage, (e.g. wall fragmentation) (fig. 6A) and, in some cases, in protoplast formation (fig. 6B) and bursting (fig. 6C). In the case of P. v e r r u c o s u m , the e n z y m a t i c t r e a t m e n t
Table 11. Effect of EDTA, NaN 3 and metal ions on the chitinolytic enzyme of V. lecanii. Relative activity (*) (%) Compound None EDTA NaN 3 KCI CaCI 2 FeS04 MgCI 2 MnCL ZnSO4
Concentration 10 mM
Concentration 100 mM
100 98 95 97 84 97 74 70 84
100 57 73 81 73 78 33 52 52
(*) Effects are shown as percentage o f the activity recorded without test c o m p o u n d taken as 100%. Results are means of three replicates with a standard deviation less than 8.0 %.
CHITINOLYTIC A C T I V I T Y OF VERTICILLIUM LECANII A3
Table III. Specificity of the chitinolytic enzyme of V. lecanii for different substrates.
Substrate
Enzyme activity ~*) (mU/rnl)
Colloidal chitin Glycol chitin Chitin from crab shells CM-cellulose Cellulose acetate Chitosan low molecular weight Chitosan medium molecular weight Chitosan high molecular weight NN'-diacetylchitobiose Xylan
340_+23 115_+6 59__.5 0 0 24_+2 37_+4 44_+3 0 25_+3
(*) The reaction mixtureconsisted of 0.5 ml 0.5% substrate in 50 mM citrate phosphatebuffer pH 4.0, exceptfor NN'-diacetyichitobiose, which was used at 5.0 mM, and 0.5 ml enzymesolution.The valuesare meansof three replicatesand standarddeviationsare shown.
295
and reduced in size (pictures not shown). In A. v e r s i c o l o r , there was no i n h i b i t i o n of spore g e r m i n a t i o n , but s o m e c e l l and m y c e l i u m damage occurred; similar results were obtained with C. c l a d o s p o r i o i d e s (pictures not shown). T h e a n t i f u n g a l a c t i v i t y w a s also t e s t e d using e n z y m e concentrations of 1 and 5 U/ml; table IV, however, reports only the effects on spore germination and hyphal length obtained with 2 U/ml. In fact, t r e a t m e n t s with 1 and 2 U/ml o f chitinase led to the same effects, while with 5 U/ml, the only difference was the c o m p l e t e inhibition of M. p l u m b e u s germination.
DISCUSSION c a u s e d i n h i b i t i o n of spore g e r m i n a t i o n that was a c c o m p a n i e d by c h a n g e s in spore morp h o l o g y w h i c h a p p e a r e d flattened, d e f l a t e d
The cell walls of m o s t p a t h o g e n i c fungi c o n t a i n c h i t i n , the d e g r a d a t i o n o f w h i c h affects g r o w t h and d i f f e r e n t i a t i o n (Poulose,
Fig. 3. Dual culture of V. lecanii and M. plumbeus. A) a= white colony, V. iecanii; b=grey colony, M. plumbeus; c=contact zone between the two microorganisms; d= overgrowth of V. lecanii on M. plumbeus. B) Detail of A. C) a=white aerial mycelium of V. lecanii overgrown on M. plumbeus: arrow shows an M. plumbeus sporangiophore with consistent size reduction; b=grey rr,ycelium of M. plumheus with black. normal sporangiophore spots (arrow).
296
M, FENICE ET AL.
reported the utilization of T. harzianum P1 as a biocontrol agent for the cool storage of carrots. The author, however, does not provide any information concerning the control mechanism at low temperature.
i¸!!!~i!~!i~!!!!!!!i!!!!!~!!!~!~i!!~!!!i!!!i~i~iJi~i!¸~i~!i!~!i~!~!~!~!~!~!~!%~!~!~!~!~!~!~!~!~!~~ i!~!~!~i!ilill !~ii! !~¸~ i!% ~¸¸¸ !~!~! Fig, 4, Dual culture o f V. lecanii and C. cladosporioides. a=white colony, V. lecanii; b=black colony, C. cladosporioides; c=contact zone between the two microorganisms; d=overgrowth of V. lecanii on C. cladosporioides.
1992). Therefore, chitinases may play an important role in the control of these organisms (Lorito et al., 1994b), In this respect, A. a l b u m , A. a l t e r n a t u m and P e n i c i l l i u m sp. (Smith et al., 1990; Malathrakis and Klironomou, 1992; Studer et al., 1992; Chet et al., 1993; Di Pietro et al., 1993; Lorito et al., 1993) have been studied. No direct correlation, however, has been established between their chitinolytic ability and mycoparasitism. The only fungal species for which this relationship has been investigated and suggested is T. harzianum and, to a lesser extent, the closely related T. (Gliocladium) virens (Di Pietro et al., 1993; Lorito et al., 1993; Chet and Inbar, 1994; Haran et al., 1994; Schirmbock et al., 1994). A major limitation to the practical utilization of T. h a r z i a n u m as a mycoparasite may be due to its inability to grow and to perform antifungal action at low temperatures (Malathrakis and Kritsotaki, 1992). Nevertheless, Tronsmo (1989) has
For its strong chitinolytic activity (Fenice et al., 1996) and its ability to grow over a wide range of temperatures (Zucconi et al., 1996), V. lecanii might in this respect be of great interest. When grown in dual cultures, the fungus showed clear mycoparasitic action against several test moulds involved in refrigerated food degradation, such as M. p l u m beus, C. cladosporioides, P. verrucosum and A. versicolor. In the case of M. plumbeus, for instance, observations under light microscope and SEM clearly showed that mycoparasitism involved the intimate invasion of its structures by V. lecanii. Similar results have been reported by Benhamou and Chet (1993) for T. harzianum grown in dual culture with Rhizoctonia solani; also in this case, SEM observations had revealed typical parasitic behaviour such as coiling of the parasite around the host, mycelial invasion and hyphal degradation. in addition to mechanical pressure (Manocha, 1987; Sahai and Manocha, 1993), the penetrating action of V. lecanii into the host mycelia seems to involve chitinolytic activity. In fact, the purified enzyme applied on the same test m i c r o r g a n i s m s ( m i c r o c u l t u r e s ) initially increased spore germination, possibly because the enzyme digested the conidial wall and then caused mycelial damage and cell lysis. On the basis of the results reported in this paper, V. lecanii can be regarded as a promising o r g a n i s m for u t i l i z a t i o n in a p p l i e d research. In fact, for its ability to grow and to release chitinase activity at low temperature, it could be useful for the biocontrol of microbial spoilage of refrigerated foods and used as a mycoparasite of phytopathogenic fungi in cold environments. Obviously, the potential of such a strain must be furth~:r investigated either with laboratory studies or by field tests.
iii~iiii!!ii!~iii~i??!!?i !il !i!~!~ii!~i!i!i!ii!~i ii!il~iiii!i!ii!ii!!i~ii!~i i!i!~~i !ili!i~ii~i!ili!~ii~ii!~~ii~IIIII ~ii~!ii!i~i!ii!ii ~i!!i!~i~iiii ; i
Fig. 5. Dual culture of V. iecanii (thin hyphae) and M. plumbeus (thick hyphae), SEM observations. 1) Initial phase of mycoparasitism: V. lecanii begins coiling around the mycelium of M. plumbeus and exerts mechanical pressure in the contact points. 2) Initial phase of mycoparasitism: V. lecanii penetrates into the host mycelium; a and b=entry and exit points of V. lecanii hyphae in the host mycelium, respectively. 3) Detail of the V. lecanii invasion of M. ph~mbeus mycelium. 4) Detail of the V. lecanii invasion of M. plumbeus conidia (a). 5) Intermediate phase of mycoparasitism: V. lecanii invades M. plumbeus (a) and degrades its cell wall in the contact points (b). The host mycelium is completely deflated. 6) Detail of 5: degradation of M. plumbeus cell wall (a) by invading hyphae of V. lecanii (arrows). 7) Advanced phase of mycoparasitism: V. lecanii completely o~crwhelms the mycelium of M. plumbeus that appears collapsed and completely deflated. 8) Advanced phase of mycoparasitism: V. lecanii invades all host .:~tructures such as the conidiophores (a). Conidia of M. plumbeus appear completely deflated (arrows).
298
M. FENICE ET AL.
B
A
~ .....
Fig. 6. Microculture of M. plumbeus treated with the purified chitinolytic enzyme (2 U/ml) of V. lecanii (observations under the light microscope, x 280). A) afconidiospore of M. plumbeus; b=conidiospore with fragmented cell wall; arrows show cell wall fragments. B) Protoplast of M. plumbeus; arrows show cell wall fragments. C) a=conidiospore of M. plumbeus; bfbursting protoplast with protoplasm extrusion (arrow).
Table IV. Microculturcs of test moulds treated with 2 U/ml of purified chitinolytic enzyme of V. lecanii.
P.v.
Tested moulds *P.v. M.p.
A.v.
*A.v.
~S0
2,8+0,3
2.8+0,3
2.6==0,2
2.6±0.2
~SI5 ILl5 %G15 t)$24 IL24 %G24
germ 27+_ 18 100 germ 63±21 100
germ 3t3± 14 100 germ 50± 15 100
2.8+_0.5 22+_ 10 85 germ 57+- 19 100
2.4+_0.4 0 0 2.0 +_0.5 0 0
*M.p.
C.c.
*C.c.
6.2+ 1.1
6.2± 1.1
17.3+_4.0 175+_88 63 germ >500 100
14.2+_5.0 30+_ 17 28 10 _+2.5 75+-44 25
4.8±0.9 7.3 ± 1.5 germ 38_+24 100 germ 40+-24 100
4.8+0.9 7.3 _+1.5 germ 43+_22 100 germ 49+_30 100
A,v.=A. versicoior ; P.v.=P. verrucosum ; M.p. = M. p l u m b e u s ; C.c. = C. cladosporioides (*indicates samples treated with the enzyme preparation); ~S0=conidiospore diameter (Bm) at time 0; t~S 15 and OS24=conidiospore diameter (Bin) at incubation time 15 and 24 h, respectively; ILl5 and IL24=hyphal length (Bm) at incubation time 15 and 24 h, respectively; %G15 and %G24=percent of germinating conidia at incubation time 15 and 24 h, respectively; germ=germinated conidia. Values represent the average of i 00 measurements_+ standard deviation.
CHITINOLYTIC A C T I V I T Y OF V E R T I C I L L I U M LECANII A3
Acknowledgements The authors gratefully acknowledge the Italian National Programme of Antarctic Research, the CRN (Centre Recherche Nestl6) and the Istituto di Patologia, University of Napoli, for kindly supplying V. cfr. lecanii A3, the strains of M. plumbeus, P. verrucosum, A. versicolor and C. cladosporioides and the two strains of Trichoderma, respectively.
Activit6 chitinolytique, ~ basse temp6rature, d'une souche antarctique (A3) de Verticillium lecanii L'activit6 chitinolytique de Verticillium cfr. lecanii A3, une souche isol6e dans le continent antarct i q u e , a 6t6 c o m p a r 6 e ~ celle de d e u x s o u c h e s s61ectionn6es de Trichoderma harzianum. Apr~s 72 h d'incubation ~ 25°C dans un milieu contenant de la chitine comme seule source de carbone, toutes les s o u c h e s ont la m ~ m e activit6 e n z y m a t i q u e (~ 2 3 0 m U / m l ) ; h 15°C, les activit6s des trois souches sont similaires; mais ~ 5°C l'activit6 de V. lecanni est environ 4 fois plus 61ev6e que celles des deux souches de T. harzianum (respectivement 203 et 54 mU/ml, pour 144 h d'incubation). La chitinase de V. lecanii s'av/~re une glycoprot6ine de 45 kDa, ie point pl 6tant ~ 4.9. La chitinase est active entre 5 et 60°C; h 5°C son activit6 relative repr6sente 5 0 % de celle not6e b. la t e m p 6 r a t u r e o p t i m a l e de 4 0 ° C V. lecanii et sa chitinase purifi6e inhibent nettement la croissance de certaines moisissures comme Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor et Penicillium verrucosum : la microscopie optique et h balayage montrent que cette inhib i t i o n de la c r o i s s a n c e est caract6ris6e p a r des 16sions myc61iennes et une lyse cellulaire. Mots-clds: Chitine, Verticillium lecanii, Trichoderma harzianum; Basses temp6ratures, Activit6 chitinolytique, Action antitbngique.
References Benhamou, N. & Chet, I. (1993), Hyphal interaction between Trichoderma harzianum and Rhizoctonia solani: ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology, 83, 10621071. Bradford, M. (1976), A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72, 248. Chet, I., Barak, Z. & Oppenheim, A. (1993), Genetic
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engineering of microorganisms for improved biocontrol activity, in "Biotechnological prospect of plant disease control" (l. Chet) (pp. 211-235). Wiley-Liss, New York. Chet, I. & Inbar, J. (1994), Biological control of fungal pathogens. Appl. Biochem. BiotechnoL, 48, 37-43. Di Pietro, A., Lorito, M., Hayes, C.K., Broadway, R.M. & Harman, G.E. (1993), Endochitinase from Gliocladium virens: isolation, characterization and synergistic antifungal activity in combination with gliotoxin. Phytopathology, 83, 308-313. Fenice, M., Selbmann, L., Di Giambattista, R., Petruccioli, M. & Federici, F. (1996), Production of extracellular chitinolytic activities by a strain of the antarctic entomogenous fungus Verticillium cfr. lecanii, in "Chitin enzymology", (R.A.A. Muzzarelli) (pp. 285-292). Atec Echzioni, Grottammare, Italy. Fenice, M., Selbmann, L., Zucconi, L. & Onofri, S. (1997), Production of extraceilular enzymes by Antarctic fungal strains. Polar Biol., 17, 275-280. Hankin, L. & Anagnostakis, S.L. (1975), The use of solid media for detection of enzyme production by fungi. Mycologia, 67, 597-607. Haran, S. Schickler, H., Oppenheim, A. & Chet, I. (1994), New components of the chitinolytic system of Trichoderma harzianum. Mycol. Res, 10, 1-6. Herman, G.E., Taylor, A.G. & Stasz, T.E. (1989), Combining effective strains of Trichoderma harzianum and solid matrix priming to improve biological seed treatment. Plant Dis., 73, 631-637. Laemmli, U.K., (1970), Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680-685. Lorito, M., Hayes, C.K., Di Pietro, A. & Harman, G.E. (1993), Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA. Curr. Genet., 24, 349-356. Lorito, M., Hayes, C.K., Di Pietro, A., Woo, S.L. & Harman, G.E. (1994a), Purification, characterization, and synergistic activity of a glucan 1,3-f3-glucosidase and an N-acetyl-13-glucoaminidase from Trichoderma harzianum. Phytopathology, 84, 398405. Lorito, M., Peterbauer, C., Hayes, C.K. & Herman, G.E. (1994b), Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination. Microbiology, 140, 623-669. Malathrakis, N.E. & Kritsotaki, O. (1992), Effect of ~ubstrate, temperature and time of application on the effectiveness of three antagonistic fungi against Botrytis cinerea, in "Recent advances in Botrytis research" (K. Verhoeff, N.E. Malathrakis & B. Williamson) (pp. 187-191). Pudoc Scientific Publishers, Wageningen. Malathrakis, N.E. & Klironomou, E.J. (1992), ~"ontrol of grey mould of tomatoes in greenhouses with fungicide and antagonists, hi "Recent advances in Botrytis research" (K. Verhoeff, N.E. Malathrakis & B. Williamson) (pp. 282-286). Pudoc Scientific Publishers, Wageningen. Manocha, M.S. (1987), Cellular and molecular aspects of fungal host-mycoparasite interaction. J. Plant Dis. Protect., 94, 431-444. Molano, J., Duran, A. & Cabib, E. (1977), A rapid cnd
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sensitive assay for chitinase using tritiated chitin. Anal Biochem., 83, 648-656. Onofri, S., Castagnola, M. & Rossi Espagnet, S. (1980), L'impiego della microscopia elettronica a scansione in micologia. Mic. ltaL, 1, 29-32. Poulose, A.J. (1992), Biotechnology and fungal control, in "Target sites of fungicide action" (W. Keller) (pp. 311-318). Boca Raton, ~ , CRC. Sahai, A.S. & Manocha, M.S. (1993), Chitinase of fungi and plants: their involvement in morphogenesis and host-parasite interaction. FEMS Microbiol. Rev., 11, 317-338. Schinnbock, M., Lofito, M., Wang, Y.-L., Hayes, C.K., Arisan-Atac, I., Scala, F., Harman, G.E. & Kubicek, C.P. (1994), Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Appl. Environ. Microbiol., 60, 4364-4370.
Smith, V.L., Wilcox, W.F. & Harman, G.E. (1990), Potential for biological Control of Phytophtora root and crown rots of apple by Trichoderma and Gliocladium spp. Phytopathology, 80, 880-885. Studer, M., Fluck, K. & Zimmermann, W. (1992), Production of chitinases by Aphanocladium album grown on crystalline and colloidal chitin. FEMS Microbiol. Lett., 99, 213-216. Tronsmo, A. (1989), Trichoderma harzianum used for biological control of storage rot on carrots. Norw. J. Agric. Sci., 3, 157-161. Zacharius, R.M., Zell, T.E., Morrison, J.H. & Woodlock, J.J. (1969), Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem., 30, 148-152. Zucconi, L., Pagano, S., Fenice, M., Selbmann, L., Tosi, S. & Onofri, S. (1996), Growth temperature preferences of fungal strains from Victoria Land. Antarctic. Polar Biol., 16, 53-61.
Res. Microbiol. 1998, 149, 289-300
Chitino|ytic activity at low temperature of an Antarctic strain (A3) of Verticillium lecanii M. Fenice, L. Selbmann, R. Di Giambattista and F. Federici ~*) Dipartimento di Agrobiologia e Agrochimica, University of Tuscia, Via S. Camillo de Lellis, 1-01100, Viterbo (Italy)
SUMMARY The chitinolytic activity of Verticillium cfr. lecanfi A3, a strain isolated from continental Antarctica, was compared to those of two selected strains of Trichoderma harzianum. After 72 h of incubation at 25 oC in media containing chitin as the sole carbon source, all strains showed the same enzyme activity (ca. 230 mU/ml); at 15°C, the levels of enzyme activity of the three strains were similar to those obtained at 250C. At 5oc, in contrast, the activity of V. lecanfi was ca. 4 times higher than those of both strains of T. harzianum (203 and 57 mU/ml, respectively; incubation time 144 h}. The chitinase of V. lecanii, purified by preparative isoelectric focusing and ion-exchange chromatography, was shown to be a glycoprotein with apparent molecular weight of 45 kDa and isoelectric point of 4.9. The enzyme was active over a broad range of temperatures (5-60"C): at 5"C, its relative activity was still 50% of that recorded at 40°C (optimal temperature). V. lecanii and its purified chitinase showed clear inhibitory effects on the growth of some test moulds such as Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor and Penicillium verrucosum: observations under the light and scanning electron microscopes revealed that growth inhibition was accompanied by myceliai damage and cell lysis.
Key-words: Chitin, Verticillium lecanii, Trichoderma harzianum; Low temperature, Chitinolytic activity, Antifungal action.
INTRODUCTION Due to undesirable side effects on the environment and human health, the use of chemical pesticides and food preservatives has been widely criticized in recent years (Chet et al., 1993; Lorito et al., 1994b). As a consequence, microorganisms have been studied in order to develop safer alternatives to chemical treatments (Chet et
Submitted November 13, 1997, accepted February 5, 1998. (*) Corresponding attthor.
al., 1993). Among fungi, Trichoderma harzianum, T. (Gliocladium) virens, Penicillium sp., A c r e m o n i u m alternatum and A p h a n o c l a d i u m album have been particularly investigated (Studer et al., 1992; Malathrakis and Kritsotaki, 1992; Malathrakis and Klironomou, 1992; Chet et al., 1993 ; Di Pietro et aL, 1993 ; Lorito et al., 1993, 1994a,b ; Schirmbock et ai., 1994). The large majority of these studies, however, have con-
290
M. FENICE ET AL.
cerned T. harzianum and Z virens, mycoparasites that produce chitinolytic enzymes involved in host cell degradation (Chet et al., 1993; Haran et aL, 1994; Lorito et al., 1994a,b; Schirmbock et al., 1994). Similar evidence is lacking for other fungal organisms. This paper deals with the chitinolytic activity at different growth temperatures of Verticillium lecanii strain A3 isolated from continental Antarctica (Fenice et al., 1996) as compared to the activity of two selected strains of T. harzianum, known for their chitinolytic activity (Harman et ai, 1989; Tronsmo, 1989). The isolation site of this fungus is an extreme terrestrial environment which can be colonized only by organisms with a high level of adaptation (Fenice et al., 1997). Thus, the characteristics of such an organism may be of interest for specific biotechnology fields, i,e. treatment of chitin-rich wastes at low temperature and/or biocontrol of phytopathogens in cold environments. In this context, the antifungal activities of V. lecanii and its purified chitinase against some spoiling agents of refrigerated foods, such as Mucor plumbeus, Penicillium verrucosum, Aspergillus versicolor and Cladosporium cladosporioides have also been investigated.
(Detroit, MI, USA). All other chemicals were of analytical grade. Microrganisms V. cfr. lecanii A3, isolated from moss samples collected in continental Antarctica, was obtained from the Dipartimento di Scienze Ambientali, University of Tuscia, Viterbo, Italy. T. harzianum P1 and T. harzianum T22 were obtained from the lstituto di Patologia Vegetale, University of Napoli "Federico 11", Portici, Naples, Italy. M. plumbeus, P. verrucosum, A. versicolor and C. cladosporioides were from the culture collection of the Life Science Department, Nestl~ Research Center (Lausanne, Switzerland). All strains are stocked in the culture collection of the Dipartimento di Agrobiologia e Agrochimica, University of Tuscia, Viterbo, Italy. During the study, the cultures were maintained on MEA at 4-6°C and subcultured every month. Chitin preparation
Colloidal chitin was prepared by treating chitin from crab shells as reported by Hankin and Anagnostakis (1975). After swelling, chitin was resuspended in water and sterilized by autoclaving (121°C, 20 min). The solid content of the sterile chitin suspension was evaluated by dry weight determinations. Glycol chitin was prepared by reacetylation of glycol chitosan using the method of Molano et al. (1977).
MATERIALS AND METHODS Culture media and growth conditions Chemicals
Chitin (from crab shells), p-nitrophenyl-B-D-Nacetylglucosaminide, p-nitrophenyl-B-D-N-N'-diacetylchitobiose, p-nitrophenyl-B-D-N-N'-N"-triacetylchitotriose, glycol chitosan and xylan were from Sigma Chemical Co. (St. Louis, MO, USA). Chitosan (low, medium and high molecular weight), cellulose acetate and CM-cellulose were from Fluka BioChemika (Buchs, CH). Malt extract agar (MEA) and potato dextrose agar (PDA) were from Oxoid (Unipath Ltd., Basingstoke, Hampshire, UK), yeast nitrogen base (YNB) was from Difco Laboratories
BM cfr. EDTA IEF MEA
= = = = =
basal medium. confrontation. ethylenediamine tetraacetate. isoelectric focusing. malt extract agar.
The basal culture medium (BM) was as follows (w/v): YNB, 1% and colloidal chitin, 1%, pH 5.5. The solid medium for dual cultures was PDA, 4 %. The liquid medium for microcultures was YNB, 1% and glucose, 1% (YG). All media were sterilized by autoclaving (121°C, 20 min). Shaken cultures
After inoculation (final concentration ca. 5.0 x 105 conidia/ml), cultures were incubated on a rotary shaker, 200 cycles/min, tor 9 days at 5, 15 and 25°C.
PDA SDS-PAGE
= =
SEM YNB
= =
potato dextrose agar. sodium dodecyl sulphatc/ptdyacrylamide gel elcctrophoresis. scanning electron microscope. yeast nitn~gen base.
CHITINOLYTIC ACTIVITY OF VERTICILLIUM LECANII A3 Samples were taken every 12-24 h and, alter centrifugafion (10,000 g, 10 min), the supernatants were used for the enzyme assay.
Dual cultures Petri dishes (10-cm diameter) filled with 20 ml of PDA were inoculated (punctiform inocula, 4 cm apart) with V. iecanii and each of the test organisms, and incubated at 25 and 5°C. In order to investigate fungal interactions and the occurrence of mycoparasitism, the mycelial structures in the contact zone of the two colonies were observed under light and scanning electron microscope (SEM).
Microcultures Thirteen l.tl of conidiospore suspensions (5 x 10 3 conidia/ml) in YG were pipetted onto sterile microscope slides and added with 13 lal of a filter sterilized solution ef purified chitinase (1.0, 2.0 and 5.0 U/ml); slides were then placed in sterile petri dishes and incubated at 25°C for 24 h in a wet thermostated room. After 15 and 24 h of incubation, the microcultures were observed under the light microscope. For the controls, sterile distilled water was used in place of the enzyme solutions.
Enzyme recovery and purification All procedures were carried out at 4°C.
Step 1: ammonium sulphate fractionated precipitation The crude enzyme solution was centrifuged (5,000 g, 10 min) and filtered (GFC membrane Whatman, UK) to remove the mycelium. The filtrate was brought to 30% saturation with ammonium sulphate, left for 2 h at 4°C and then centrifuged (5,000 g, 10 min). The supernatant was then brought to 75 % saturation with the same salt and left overnight at 4°C. The precipitate formed was collected by centrifugation (8,000 g, 15 min), dissolved in 15 ml of 5 mM citrate-phosphate buffer pH 5.0 and dialysed overnight against several volumes of the same buffer. The final enzyme solution, which contained ca. 90% of the initial chitinolytic activity, was concentrated by lyophilization. The lyophilized material had an activity of 2.3 U/mg of protein.
Step 2: preparative isoelectric fi~cusing (Rotofor) After resuspension in distilled water, the ]y~ philized enzyme from step 1 was applied t~ a
291
"Rotofor" apparatus (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's directions, and R o t o l y t e s ( B i o - R a d Laboratories) (range 4.5-6.1) at a gradient of 4.5-5.7 were used. Peak fractions were collected, pooled and concentrated by centrifugation (6,000 g, 10 min) with a "Centricon 10000" membrane system (Amicon, USA).
Step 3: ion-exchange column chromatography The dialysed enzyme from step 2 was directly applied to an "Econo-pack Q" cartridge (Q-sepharose) (Bio-Rad Laboratories) equilibrated with 20 mM tris-HCl buffer pH 8.0 and eluted with a 3step gradient of the same buffer containing 0-1 M NaCI at a flow rate of 1 ml/min. Fractions (2.0 ml each) containing the chitinolytic activity were pooled and concentrated as above.
Enzyme characterization The effect of pH on the enzyme activity was determined by varying the pH of reaction mixtures using 50 mM glycine/HCl buffer (pH 1.0-3.0), 50 mM citrate phosphate buffer (pH 3.5-7.0) and 50 mM phosphate buffer (pH 7.0-8.0). The denaturing effects of pH were investigated by incubating the enzyme solution for 1 h at 25°C in the range of pH values from 3.0 to 6.0. The effect of temperature on the enzyme activity was determined at pH 4.0 in the range of temperatures from 5 to 80°C. Temperature stability was evaluated by incubating the enzyme solution at 40°C and pH 4.0 for 10 h: samples were taken every hour and the residual enzyme activity determined. The effects of metal ions such as K +, Ca ++, Fe ++, Mg ++, Mn +÷, Zn ++, sodium azide and EDTA on the apparent enzyme activity were determined after preincubation (37°C, 30 min) of the enzyme solution with 10 and 100 mM of the above compounds.
Electrophoresis Sodium dodecyl sulphate/polyacry]amide gel electrophoresis (SDS-PAGE) was used to check the purity of protein and to determine the molecular mass of the purified enzyme under denaturing conditions using a 12.0% polyacrylamide gel, according to Laemmli (1970). After electrophoresis, the gels were stained with Coomassie brilliant blue R-250 (Pharmacia, Sweden) or by the periodic acid Schiff reagent for glycoproteins (Zacharius eta/., 1969). Molecular markers (low range) were from Bio-Rad Laboratories (CA).
292
M. F E N I C E E T AL.
Isoelectric focusing (IEF) The isoelectric point (pl) of the purified chitinase was determined using a "Multiphor II" cell (Pharmacia, Sweden) and a precasted polyacrylamide gel "Pagplate" (Pharmacia, Sweden) with ampholine pH range 4.0-6.5. Broad-range (3.5-10) IEF gel protein calibration kits were from Pharmacia (Sweden); stainings for protein were as described for SDSPAGE,
V. lecanii reached the same level of chitinolytic activity (228 mU/ml) at the same incubation time (72 h); after this time, however, its enzyme activity began to decrease. The maximal activity of T. h a r z i a n u m P I was c o n s i d e r a b l y lower (121 mU;ml) and was reached only after 120 h of incubation (fig. IA). At 15°C, the three strains showed the same levels of activity as above, but production was delayed ca. 48 h (fig. IB). At 5°C, V. lecanii reached a level of enzyme activity
Protein estimation Protein concentration was estimated spectrophotometrically at 595 nm using bovine serum albumin as a standard protein, by the Bio-Rad Laboratories (CA) procedure (Bradford, 1976).
25O
A
200
Enzyme assay
150
Chitinolytic activity was determined as previously reported (Fenice et al., 1996) by measuring the amount of reducing sugars liberated from colloidal chitin by the enzyme activity; N-acetyl-Dglucosamine was used for the standard curve. Under the assay conditions, one unit (U) of enzyme activity was defined as the amount of enzyme which released 1 lamol of N-D-acetylglucosamine per milliliter per minute.
!00
50 0
|
|
I
i
250 2(X) 150
Scanning electron microscopy Samples from different places of petri dishes containing dual cultures were cut with a sharp lancet and treated as reported by Onofri et al. (1980). A "Jeol 5002 JSM" (Jeol, Japan) SEM was used.
al
5o
0
250
RESULTS
Chitinolytic act.~vity of V. lecanii at different growth temperatures V. lecanii was grown at different temperatures (5, 15 and 25°C) and its chitinolytic activity compared to those of two selected strains of T. harzianum (T. harzianum PI and Z harzianum T22); results are reported in figure 1.
At 25°C, T. harzianum T22 reached its maximal activity (230 mU/ml) after 72 h of incubation, and the activity remained almost stable for 72 more hours to decrease markedly thereafter.
I
.....
1
I
c
200
150
100
50
v
24
48
72
9b
120
144
168
192
216
Time (hours)
Fig. 1. Chitinolytic activity of V. lecanii (0), T. harziamlm PI (V) and T. harzianmn T22 (O) grown at 25 (A), 15 (B) and 5°C (C). Values represent the average of three experiments and error bars indicate standard deviations.
CHITINOLYTIC ACTIVITY OF VERTICILLIUM LECANI] A3
(203 mU/ml after 144 h of incubation) very close to that obtained at 25°C. T. harzianum T22 and T. harzianum PI, on the contrary, showed much lower levels of activity (22 and 57 mU/rnl, respectively), and the same fermentation time (fig. 1C).
Enzyme purification and charactedzation The crude enzyme from V. lecanff culture was fractionated by preparative IEF (Rotofor) and ion-exchange chromatography (Q-sepharose). After Rotofor separation, the chitinolytic activity was found in fractions with pH about 5.0. These fractions were combined and applied to ion exchange, eluted within the gradient (ca. 20% of 1M NaCI) and recovered as a single peak. The recovery yield was 14% of the initial enzyme activity (table I). After ion-exchange chromatography, fractions showing chitinolytic activity were pooled and submitted to SDS-PAGE: a single band was obtained, with apparent molecular weight of 45 kDa (fig. 2). After staining with the periodic acid Schiff reagent (Zacharius et al., 1969), the chitinase appeared to be a glycoprotein having an isoelectric point of 4.9 (data not shown). The optimal pH for activity was 4.0; at pH 3.5 and 4.5 the enzyme retained about 53 and 66 % of the activity shown at pH 4.0, respectively. The denaturing effect of pH was tested by incubating the enzyme solution for lh at 25°C in the pH range 3.0-6.0: the enzyme was most stable at pH 5.0. The purified chitinase was active over the temperature range 5-60°C with a maximum at 40°C; at 5°C, the enzyme retained 50% of the activity shown at 40°C. After 1, 4 and 8 h of incubation at optimal pH and temperature, the
293
enzyme maintained 80, 50 and 5 % of the initial activity, respectively. The effects of different metal ions, EDTA and NaN 3 on the enzyme activity are reported in table lI. At 10 mM, K +, Fe ++, NaN 3 and EDTA did not produce any inhibitory effects, while the other substances caused 30 % inhibition. At 100 mM, the inhibitory effects were more evident: Zn ÷+, ML ~+ and EDTA caused an activity reduction of about 50 % and with Mg ++ the residual activity was about 30%. The chitinolytic enzyme of V lecanii hydroiysed colloidal a~,'7 glycol chitin more easily than other chitins (table III). There was no activity on either the celluloses or NN'-diacetylchitobiose; xylan and chitosans were degraded to a minor extent.
Ant~ungal activity of V. lecanii and its purified chitinase
V. lecanii and its purified chitinase showed clear inhibitory effects on a series of test moulds (M. plumbeus, C. cladosporioides, P verrucosum and A. versicolor) (table IV and figs. 3-6).
Figure 3A shows the appearance of a dual culture of V. lecanii and M. plumbeus after 15 days of incubation at 25°C. An area of lysis of the mycelium of M. plumbeus was present in the contact zone between the two microorganisms (centre of the plate) (fig. 3A). Also, V. lecanii (white aerial mycelium) almost completely overgrew M. plumbeus (light grey mycelium with black spots of sporangia shown in figure 3B). Furthermore, some structures, such as the sporangiophores, appeared to be damaged, with a considerable reduction in size (fig. 3C).
Table I. Purification of the chitinolytic activity of V. lecanii.
Step
Protein (rag)
Chitinase (U)
Sp. activity (U/mg)
Yield (%)
Purification (fold)
(NH4)2SO4 Rotofor Q-sepharose
68.3 9.18 0.043
204.4 89.1 28.0
2.99 9.70 65! .2
100 44 14
1 3 218
294
M. FENICE ET AL.
97A
66,2
Q 31
2t,S
t
2
$
KDa
Fig. 2. SDS-PAGE (12%) electrophoresis of the purified chitinolytic enzyme of V. lecanii. Lane 1= ion exchange (Q-sepharose); lane 2 = Rotofor; lane 3=ammonium sulphate precipitate. Molecular weight markers were bovine albumin (66 kDa), egg albumin (45 kDa), carbonic anhydrase (29 kDa) and trypsin inhibitor (20 kDa). Gel was stained with Coomassie blue.
Figure 4 shows a dual culture of V. lecanii and C. cladosporioides: the latter was almost overwhelmed by the mycelial growth of the former. Similar results were obtained with dual cultures of V. lecanii and P. verrucosum or A. versicolor (pictures not shown).
Samples were collected from the interaction region of dual cultures of V. lecanii and M. p l u m b e u s and o b s e r v e d u n d e r the SEM (fig. 5). V. lecanii established a very close contact with M. plumbeus: it coiled around the host mycelia (fig. 5-1) and penetrated into its hyphae (figs. 5-2 and 5-3), sporangia and sporangiospores (figs. 5-4 and 5-8). The mycoparasitic action of V. lecanii on M. plumbeus produced hyphal loss of turgor and degradation (figs. 5-5, 5-6 and 5-7) and spore deflating (fig. 5-8). The same results were obtained when dual cultures were incubated at 5°C; at this temperature, however, 30 days of incubation were necessary to observe the mycoparasitic effect (data not shown). Microcultures of the above test organisms, treated with 2 U/ml of the purified chitinase from V. lecanii, showed inhibition of spore germination, mycelium vacuolization and lysis, cell wall disruption, protoplast formation and bursting. M. p l u m b e u s , after 15 h of i n c u b a t i o n , showed r e d u c e d or no spore g e r m i n a t i o n . After 24 h, an inhibitory effect began to be evident that consisted in m a r k e d cell wall damage, (e.g. wall fragmentation) (fig. 6A) and, in some cases, in protoplast formation (fig. 6B) and bursting (fig. 6C). In the case of P. v e r r u c o s u m , the e n z y m a t i c t r e a t m e n t
Table 11. Effect of EDTA, NaN 3 and metal ions on the chitinolytic enzyme of V. lecanii. Relative activity (*) (%) Compound None EDTA NaN 3 KCI CaCI 2 FeS04 MgCI 2 MnCL ZnSO4
Concentration 10 mM
Concentration 100 mM
100 98 95 97 84 97 74 70 84
100 57 73 81 73 78 33 52 52
(*) Effects are shown as percentage o f the activity recorded without test c o m p o u n d taken as 100%. Results are means of three replicates with a standard deviation less than 8.0 %.
CHITINOLYTIC A C T I V I T Y OF VERTICILLIUM LECANII A3
Table III. Specificity of the chitinolytic enzyme of V. lecanii for different substrates.
Substrate
Enzyme activity ~*) (mU/rnl)
Colloidal chitin Glycol chitin Chitin from crab shells CM-cellulose Cellulose acetate Chitosan low molecular weight Chitosan medium molecular weight Chitosan high molecular weight NN'-diacetylchitobiose Xylan
340_+23 115_+6 59__.5 0 0 24_+2 37_+4 44_+3 0 25_+3
(*) The reaction mixtureconsisted of 0.5 ml 0.5% substrate in 50 mM citrate phosphatebuffer pH 4.0, exceptfor NN'-diacetyichitobiose, which was used at 5.0 mM, and 0.5 ml enzymesolution.The valuesare meansof three replicatesand standarddeviationsare shown.
295
and reduced in size (pictures not shown). In A. v e r s i c o l o r , there was no i n h i b i t i o n of spore g e r m i n a t i o n , but s o m e c e l l and m y c e l i u m damage occurred; similar results were obtained with C. c l a d o s p o r i o i d e s (pictures not shown). T h e a n t i f u n g a l a c t i v i t y w a s also t e s t e d using e n z y m e concentrations of 1 and 5 U/ml; table IV, however, reports only the effects on spore germination and hyphal length obtained with 2 U/ml. In fact, t r e a t m e n t s with 1 and 2 U/ml o f chitinase led to the same effects, while with 5 U/ml, the only difference was the c o m p l e t e inhibition of M. p l u m b e u s germination.
DISCUSSION c a u s e d i n h i b i t i o n of spore g e r m i n a t i o n that was a c c o m p a n i e d by c h a n g e s in spore morp h o l o g y w h i c h a p p e a r e d flattened, d e f l a t e d
The cell walls of m o s t p a t h o g e n i c fungi c o n t a i n c h i t i n , the d e g r a d a t i o n o f w h i c h affects g r o w t h and d i f f e r e n t i a t i o n (Poulose,
Fig. 3. Dual culture of V. lecanii and M. plumbeus. A) a= white colony, V. iecanii; b=grey colony, M. plumbeus; c=contact zone between the two microorganisms; d= overgrowth of V. lecanii on M. plumbeus. B) Detail of A. C) a=white aerial mycelium of V. lecanii overgrown on M. plumbeus: arrow shows an M. plumbeus sporangiophore with consistent size reduction; b=grey rr,ycelium of M. plumheus with black. normal sporangiophore spots (arrow).
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reported the utilization of T. harzianum P1 as a biocontrol agent for the cool storage of carrots. The author, however, does not provide any information concerning the control mechanism at low temperature.
i¸!!!~i!~!i~!!!!!!!i!!!!!~!!!~!~i!!~!!!i!!!i~i~iJi~i!¸~i~!i!~!i~!~!~!~!~!~!~!%~!~!~!~!~!~!~!~!~!~~ i!~!~!~i!ilill !~ii! !~¸~ i!% ~¸¸¸ !~!~! Fig, 4, Dual culture o f V. lecanii and C. cladosporioides. a=white colony, V. lecanii; b=black colony, C. cladosporioides; c=contact zone between the two microorganisms; d=overgrowth of V. lecanii on C. cladosporioides.
1992). Therefore, chitinases may play an important role in the control of these organisms (Lorito et al., 1994b), In this respect, A. a l b u m , A. a l t e r n a t u m and P e n i c i l l i u m sp. (Smith et al., 1990; Malathrakis and Klironomou, 1992; Studer et al., 1992; Chet et al., 1993; Di Pietro et al., 1993; Lorito et al., 1993) have been studied. No direct correlation, however, has been established between their chitinolytic ability and mycoparasitism. The only fungal species for which this relationship has been investigated and suggested is T. harzianum and, to a lesser extent, the closely related T. (Gliocladium) virens (Di Pietro et al., 1993; Lorito et al., 1993; Chet and Inbar, 1994; Haran et al., 1994; Schirmbock et al., 1994). A major limitation to the practical utilization of T. h a r z i a n u m as a mycoparasite may be due to its inability to grow and to perform antifungal action at low temperatures (Malathrakis and Kritsotaki, 1992). Nevertheless, Tronsmo (1989) has
For its strong chitinolytic activity (Fenice et al., 1996) and its ability to grow over a wide range of temperatures (Zucconi et al., 1996), V. lecanii might in this respect be of great interest. When grown in dual cultures, the fungus showed clear mycoparasitic action against several test moulds involved in refrigerated food degradation, such as M. p l u m beus, C. cladosporioides, P. verrucosum and A. versicolor. In the case of M. plumbeus, for instance, observations under light microscope and SEM clearly showed that mycoparasitism involved the intimate invasion of its structures by V. lecanii. Similar results have been reported by Benhamou and Chet (1993) for T. harzianum grown in dual culture with Rhizoctonia solani; also in this case, SEM observations had revealed typical parasitic behaviour such as coiling of the parasite around the host, mycelial invasion and hyphal degradation. in addition to mechanical pressure (Manocha, 1987; Sahai and Manocha, 1993), the penetrating action of V. lecanii into the host mycelia seems to involve chitinolytic activity. In fact, the purified enzyme applied on the same test m i c r o r g a n i s m s ( m i c r o c u l t u r e s ) initially increased spore germination, possibly because the enzyme digested the conidial wall and then caused mycelial damage and cell lysis. On the basis of the results reported in this paper, V. lecanii can be regarded as a promising o r g a n i s m for u t i l i z a t i o n in a p p l i e d research. In fact, for its ability to grow and to release chitinase activity at low temperature, it could be useful for the biocontrol of microbial spoilage of refrigerated foods and used as a mycoparasite of phytopathogenic fungi in cold environments. Obviously, the potential of such a strain must be furth~:r investigated either with laboratory studies or by field tests.
iii~iiii!!ii!~iii~i??!!?i !il !i!~!~ii!~i!i!i!ii!~i ii!il~iiii!i!ii!ii!!i~ii!~i i!i!~~i !ili!i~ii~i!ili!~ii~ii!~~ii~IIIII ~ii~!ii!i~i!ii!ii ~i!!i!~i~iiii ; i
Fig. 5. Dual culture of V. iecanii (thin hyphae) and M. plumbeus (thick hyphae), SEM observations. 1) Initial phase of mycoparasitism: V. lecanii begins coiling around the mycelium of M. plumbeus and exerts mechanical pressure in the contact points. 2) Initial phase of mycoparasitism: V. lecanii penetrates into the host mycelium; a and b=entry and exit points of V. lecanii hyphae in the host mycelium, respectively. 3) Detail of the V. lecanii invasion of M. ph~mbeus mycelium. 4) Detail of the V. lecanii invasion of M. plumbeus conidia (a). 5) Intermediate phase of mycoparasitism: V. lecanii invades M. plumbeus (a) and degrades its cell wall in the contact points (b). The host mycelium is completely deflated. 6) Detail of 5: degradation of M. plumbeus cell wall (a) by invading hyphae of V. lecanii (arrows). 7) Advanced phase of mycoparasitism: V. lecanii completely o~crwhelms the mycelium of M. plumbeus that appears collapsed and completely deflated. 8) Advanced phase of mycoparasitism: V. lecanii invades all host .:~tructures such as the conidiophores (a). Conidia of M. plumbeus appear completely deflated (arrows).
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B
A
~ .....
Fig. 6. Microculture of M. plumbeus treated with the purified chitinolytic enzyme (2 U/ml) of V. lecanii (observations under the light microscope, x 280). A) afconidiospore of M. plumbeus; b=conidiospore with fragmented cell wall; arrows show cell wall fragments. B) Protoplast of M. plumbeus; arrows show cell wall fragments. C) a=conidiospore of M. plumbeus; bfbursting protoplast with protoplasm extrusion (arrow).
Table IV. Microculturcs of test moulds treated with 2 U/ml of purified chitinolytic enzyme of V. lecanii.
P.v.
Tested moulds *P.v. M.p.
A.v.
*A.v.
~S0
2,8+0,3
2.8+0,3
2.6==0,2
2.6±0.2
~SI5 ILl5 %G15 t)$24 IL24 %G24
germ 27+_ 18 100 germ 63±21 100
germ 3t3± 14 100 germ 50± 15 100
2.8+_0.5 22+_ 10 85 germ 57+- 19 100
2.4+_0.4 0 0 2.0 +_0.5 0 0
*M.p.
C.c.
*C.c.
6.2+ 1.1
6.2± 1.1
17.3+_4.0 175+_88 63 germ >500 100
14.2+_5.0 30+_ 17 28 10 _+2.5 75+-44 25
4.8±0.9 7.3 ± 1.5 germ 38_+24 100 germ 40+-24 100
4.8+0.9 7.3 _+1.5 germ 43+_22 100 germ 49+_30 100
A,v.=A. versicoior ; P.v.=P. verrucosum ; M.p. = M. p l u m b e u s ; C.c. = C. cladosporioides (*indicates samples treated with the enzyme preparation); ~S0=conidiospore diameter (Bm) at time 0; t~S 15 and OS24=conidiospore diameter (Bin) at incubation time 15 and 24 h, respectively; ILl5 and IL24=hyphal length (Bm) at incubation time 15 and 24 h, respectively; %G15 and %G24=percent of germinating conidia at incubation time 15 and 24 h, respectively; germ=germinated conidia. Values represent the average of i 00 measurements_+ standard deviation.
CHITINOLYTIC A C T I V I T Y OF V E R T I C I L L I U M LECANII A3
Acknowledgements The authors gratefully acknowledge the Italian National Programme of Antarctic Research, the CRN (Centre Recherche Nestl6) and the Istituto di Patologia, University of Napoli, for kindly supplying V. cfr. lecanii A3, the strains of M. plumbeus, P. verrucosum, A. versicolor and C. cladosporioides and the two strains of Trichoderma, respectively.
Activit6 chitinolytique, ~ basse temp6rature, d'une souche antarctique (A3) de Verticillium lecanii L'activit6 chitinolytique de Verticillium cfr. lecanii A3, une souche isol6e dans le continent antarct i q u e , a 6t6 c o m p a r 6 e ~ celle de d e u x s o u c h e s s61ectionn6es de Trichoderma harzianum. Apr~s 72 h d'incubation ~ 25°C dans un milieu contenant de la chitine comme seule source de carbone, toutes les s o u c h e s ont la m ~ m e activit6 e n z y m a t i q u e (~ 2 3 0 m U / m l ) ; h 15°C, les activit6s des trois souches sont similaires; mais ~ 5°C l'activit6 de V. lecanni est environ 4 fois plus 61ev6e que celles des deux souches de T. harzianum (respectivement 203 et 54 mU/ml, pour 144 h d'incubation). La chitinase de V. lecanii s'av/~re une glycoprot6ine de 45 kDa, ie point pl 6tant ~ 4.9. La chitinase est active entre 5 et 60°C; h 5°C son activit6 relative repr6sente 5 0 % de celle not6e b. la t e m p 6 r a t u r e o p t i m a l e de 4 0 ° C V. lecanii et sa chitinase purifi6e inhibent nettement la croissance de certaines moisissures comme Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor et Penicillium verrucosum : la microscopie optique et h balayage montrent que cette inhib i t i o n de la c r o i s s a n c e est caract6ris6e p a r des 16sions myc61iennes et une lyse cellulaire. Mots-clds: Chitine, Verticillium lecanii, Trichoderma harzianum; Basses temp6ratures, Activit6 chitinolytique, Action antitbngique.
References Benhamou, N. & Chet, I. (1993), Hyphal interaction between Trichoderma harzianum and Rhizoctonia solani: ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology, 83, 10621071. Bradford, M. (1976), A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72, 248. Chet, I., Barak, Z. & Oppenheim, A. (1993), Genetic
299
engineering of microorganisms for improved biocontrol activity, in "Biotechnological prospect of plant disease control" (l. Chet) (pp. 211-235). Wiley-Liss, New York. Chet, I. & Inbar, J. (1994), Biological control of fungal pathogens. Appl. Biochem. BiotechnoL, 48, 37-43. Di Pietro, A., Lorito, M., Hayes, C.K., Broadway, R.M. & Harman, G.E. (1993), Endochitinase from Gliocladium virens: isolation, characterization and synergistic antifungal activity in combination with gliotoxin. Phytopathology, 83, 308-313. Fenice, M., Selbmann, L., Di Giambattista, R., Petruccioli, M. & Federici, F. (1996), Production of extracellular chitinolytic activities by a strain of the antarctic entomogenous fungus Verticillium cfr. lecanii, in "Chitin enzymology", (R.A.A. Muzzarelli) (pp. 285-292). Atec Echzioni, Grottammare, Italy. Fenice, M., Selbmann, L., Zucconi, L. & Onofri, S. (1997), Production of extraceilular enzymes by Antarctic fungal strains. Polar Biol., 17, 275-280. Hankin, L. & Anagnostakis, S.L. (1975), The use of solid media for detection of enzyme production by fungi. Mycologia, 67, 597-607. Haran, S. Schickler, H., Oppenheim, A. & Chet, I. (1994), New components of the chitinolytic system of Trichoderma harzianum. Mycol. Res, 10, 1-6. Herman, G.E., Taylor, A.G. & Stasz, T.E. (1989), Combining effective strains of Trichoderma harzianum and solid matrix priming to improve biological seed treatment. Plant Dis., 73, 631-637. Laemmli, U.K., (1970), Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680-685. Lorito, M., Hayes, C.K., Di Pietro, A. & Harman, G.E. (1993), Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA. Curr. Genet., 24, 349-356. Lorito, M., Hayes, C.K., Di Pietro, A., Woo, S.L. & Harman, G.E. (1994a), Purification, characterization, and synergistic activity of a glucan 1,3-f3-glucosidase and an N-acetyl-13-glucoaminidase from Trichoderma harzianum. Phytopathology, 84, 398405. Lorito, M., Peterbauer, C., Hayes, C.K. & Herman, G.E. (1994b), Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination. Microbiology, 140, 623-669. Malathrakis, N.E. & Kritsotaki, O. (1992), Effect of ~ubstrate, temperature and time of application on the effectiveness of three antagonistic fungi against Botrytis cinerea, in "Recent advances in Botrytis research" (K. Verhoeff, N.E. Malathrakis & B. Williamson) (pp. 187-191). Pudoc Scientific Publishers, Wageningen. Malathrakis, N.E. & Klironomou, E.J. (1992), ~"ontrol of grey mould of tomatoes in greenhouses with fungicide and antagonists, hi "Recent advances in Botrytis research" (K. Verhoeff, N.E. Malathrakis & B. Williamson) (pp. 282-286). Pudoc Scientific Publishers, Wageningen. Manocha, M.S. (1987), Cellular and molecular aspects of fungal host-mycoparasite interaction. J. Plant Dis. Protect., 94, 431-444. Molano, J., Duran, A. & Cabib, E. (1977), A rapid cnd
300
M. F E N I C E E T AL.
sensitive assay for chitinase using tritiated chitin. Anal Biochem., 83, 648-656. Onofri, S., Castagnola, M. & Rossi Espagnet, S. (1980), L'impiego della microscopia elettronica a scansione in micologia. Mic. ltaL, 1, 29-32. Poulose, A.J. (1992), Biotechnology and fungal control, in "Target sites of fungicide action" (W. Keller) (pp. 311-318). Boca Raton, ~ , CRC. Sahai, A.S. & Manocha, M.S. (1993), Chitinase of fungi and plants: their involvement in morphogenesis and host-parasite interaction. FEMS Microbiol. Rev., 11, 317-338. Schinnbock, M., Lofito, M., Wang, Y.-L., Hayes, C.K., Arisan-Atac, I., Scala, F., Harman, G.E. & Kubicek, C.P. (1994), Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Appl. Environ. Microbiol., 60, 4364-4370.
Smith, V.L., Wilcox, W.F. & Harman, G.E. (1990), Potential for biological Control of Phytophtora root and crown rots of apple by Trichoderma and Gliocladium spp. Phytopathology, 80, 880-885. Studer, M., Fluck, K. & Zimmermann, W. (1992), Production of chitinases by Aphanocladium album grown on crystalline and colloidal chitin. FEMS Microbiol. Lett., 99, 213-216. Tronsmo, A. (1989), Trichoderma harzianum used for biological control of storage rot on carrots. Norw. J. Agric. Sci., 3, 157-161. Zacharius, R.M., Zell, T.E., Morrison, J.H. & Woodlock, J.J. (1969), Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem., 30, 148-152. Zucconi, L., Pagano, S., Fenice, M., Selbmann, L., Tosi, S. & Onofri, S. (1996), Growth temperature preferences of fungal strains from Victoria Land. Antarctic. Polar Biol., 16, 53-61.