STUDIA FORESTALIA SUECICA

The degradation of and the production of , xylanase, mannanase and by wood - attacking microfungi

Cellulosanedbrytning och produktion av cellulas, xylanas, mannanas och arnylas hos vedangripande mikrosvampar.

THOMAS NILSSON Department of Forest Products

SKOGSHOGSKOLAN ROYAL COLLEGE OF FORESTRY STOCKHOLM Abstract

Thirty-six species of wood-inhabiting microfungi have been assayed for cellulase, xylanase, mannanase and amylase activity by the use of different test methods. The wood-degrading capabilities of the test organisms were previously known. Three of the species were unable to degrade wood. The other species represented three different degradation patterns, viz. I) formation of soft rot cavities (Type 1 attack); 2) erosion of the wood cell walls (Type 2 attack);and 3) simultaneous Type 1 and Type 2 attack.

'With one exception, all species were able to degrade starch. The production of the enzymes cellulase, xylanase and mannanase was demonstrated for twenty of the wood-degrading species. Five of the wood-degrading species, which all produced soft rot cavities in birch wood, failed to exhibit any of the enzyme activities. The remaining wood-degrading species could be shown to produce one or two of the enzymes. One of the species which was unable to degrade birch wood produced xylanase. Cellulase and mannanase were not produced by any of these species.

Twelve of the wood-degrading species appeared unable to degrade pure cellulose substrates when grown on agar media or in liquid cultures. Their only exhibition of cellulolytic activity was found in solid birch wood, where they formed soft rot cavities. These species have been referred to as "non-cellulolytic" soft rot fungi. Various explanations of the anomalous behaviour of these species are discussed.

Ms received 27th February, 1974

Allmanna Forlaget ISBN 91-38-01913-2 Tryck: Tryckindustri AB, Solna, 1974 Contents

1 Introduction ...... 5

2 Materials and methods ...... 6 2.1 Organisms ...... 6 2.2 Substrates ...... 6 2.3 Assay of cellulase, xylanase, mannanase and amylase with the RautelaCowling technique 6 2.4 Cultivation on cellulose agar plates and assay of the cellulase produced . . . . 7 2.5 Assay of cellulase and xylanase present in wood blocks attacked by the test organisms 9 2.6 Cultivation in liquid media. Weight losses of cellulosic substrates and assays of cellu- lase and xylanase ...... 9 3 Results ...... 3.1 Type of attack produced in birch wood . 3.2 Clearing produced in agar columns with cellulose, xylan, glucomannan and starch (Rautela-Cowling technique) . . . . . 3.3 Growth and cellulase production on cellu- lose agar plates ...... 3.4 Presence of cellulase and xylanase in birch wood attacked by the test organisms . . 3.5 Growth onmedium EP and B-VII-L in liquid cultures with glucose as source of carbon 3.6 Degradation of cellulose N, Avicel and cot- ton and the production of cellulase and xy- lanase on these substrates in liquid cultures 3.7 Degradation of birch wood meal and the production of cellulase and xylanase on this substrate in liquid cultures . . . 3.8 Degradation of cotton and jute fibres and the production of cellulase and xylanase on these substrates in liquid cultures . . 3.9 Effect of various amounts of glucose added to medium EP on the production of cellulase and xylanase ...... 3.10 Comparison of the results obtained with different test methods ......

4 Discussion ...... 22 4.1 Wood degradation and enzyme production 22 4.2 Test methods and results ...... 24 4.3 The anomalous behaviour of the "non- cellulolytic" soft rot fungi ...... 31

Summary ...... 35 Acknowledgements ...... 37 Sammanfattning ...... 3 8 References ...... 40

Tables ...... 43 1 Introduction

In a previous paper (Nilsson 1973) the method for cellulolytic activity mentioned celluloly tic activity of 160 species of micro- above had been used. fungi was compared with their ability to degrade birch wood (Betula verrucosa In order to investigate whether this anoma- Ehrh.). The Rautela and Cowling (1966) lous behaviour is a consistent feature of this method was used for assays of celluloly- group of fungi or whether it is an effect due tic activity. With this method cellulolytic to the test method used for assaying cellulo- activity is measured as the depth of clearing lytic activity, further studies have been of cellulose beneath fungal cultures growing carried out with other assay methods. The on the top of agar columns in test tubes. The studies have also been extended to assays of ability to degrade birch wood was deter- xylanase, mannanase and amylase. It is likely mined by microscopic observations on sec- that xylanase and mannanase are involved in tions from wood which had been attacked. the degradation of wood and it was intended Loss in weight of the birch wood was also to examine whether fungi which had failed determined for several of the species. to show cellulolytic activity also would fail to exhibit xylanase and mannanase activities. When using the decay method employed Amylase activity was assayed only for the (Nilsson 1973), it was found that the micro- purposes of comparison. fungi could be classified into four groups with respect to their type of attack in birch Thirty-six different species of microfungi wood: 1) fungi producing no attack, or were selected from our culture collections producing no other attack than small bore for these studies. Their type of wood attack holes through the cell walls; 2) fungi produ- was known and the species were selected so cing only soft rot cavities (Type 1 attack); that representatives from all of the four 3) fungi producing only a type of erosion of groups of the fungi mentioned above were the cell walls (Type 2 attack); and 4) fungi included. producing both soft rot cavities and erosion. The objects of the present investigation were A comparison of the results from the assays two; 1) to demonstrate the production of of cellulolytic activity and the results from cellulase, xylanase and mannanase, enzymes the wood decay tests showed that there was which a priori were assumed to be produced a good correlation between the actual clear- by the wood-degrading species, and 2) to ing of cellulose and the ability to degrade obtain information about the conditions birch wood. Of the 109 species which under which these enzymes are produced in produced clearing of cellulose, 105 (96.3 %) order to achieve a better understanding of were able to degrade birch wood. However, the mechanisms of wood degradation. No several of the species tested (9.4 %), which specific studies of the activities of the were able to produce soft rot cavities in the various enzymes were attempted. The pur- wood, failed to produce any clearing of pose was merely to demonstrate the presen- cellulose. They would thus have been consi- ce or absence of the various enzymes by the dered as non-cellulolytic if only the assay use of different test methods. 2 Material and methods

2.1 Organisms chloric acid according to a method described by Bose ( 1963). Thirty-six different species of microfungi were studied. All species are listed in Table 1. Cotton wool. Chemically pure (Kebo AB). Data on isolation, type of wood attack, weight losses of wood and cellulolytic activity Cellulose N (Nattraby cellstoff). This cellulo- can be obtained from a previous paper se is a commercial product prepared from (Nilsson 1973) for all but four of the equal amounts of bleached pine sulphate species, viz. Ceratocystis albida (Mathiesen- pulp and bleached pine sulphite pulp. Accor- Kaarik) Hunt, Ceratocystis stenoceras (Ro- ding to the manufacturer (Molnlycke AB) bak) C. Moreau, an unidentified Cladospo- this cellulose contains only small amounts of rium species, tentatively called Cladospo- hemicellulose. rium sp. A, and an unidentified imperfect called Fungus D. Fungus D is very Jute fibres. From Corchorus olitorius (red common in in chip piles and preser- jute). Received from Dr. N.J. Poole at the vative treated poles. The strain used here was School of Agriculture, Aberdeen. isolated 1968 from spruce pulpwood chips. Ceratocystis albida (strain B-23) was isola- Birch wood meal. 80 mesh. Prepared from ted 1952 by Dr. A. Kaarik from galleries of Betula verrucosa. Pissodes pini in a pine log. Ceratocystis stenoceras (strain B-104) was obtained Larch xylan. (Koch-Light Laboratories from "Centraalbureau voor Schimmelcultu- Ltd.). res" in Baarn, and Cladosporium sp. A (strain SP78-4) was isolated 1971 from a Glucomannan. Prepared from Pinus silvestris pine foundation pile. at Swedish Forest Products Research Labo- ratory. The preparation contained 78.2 per- cent mannose, 17.8 percent glucose, 1.4 percent galactose, 1.4 percent xylose and 1.2 2.2 Substrates percent arabinose.

Avicel. A microcrystalline cellulose prepara- Starch. Soluble starch. (E. Merck, Darm- tion. Average particle size 38 ,U (Kebo AB). stadt).

Walseth cellulose. Cellulose swollen in 85 percent o-phosphoric acid. Prepared from cellulose powder (Whatman CF 11) accor- 2.3 Assay of cellulase, xylanase, mannanase ding to the description by Rautela and and amylase with the Rautela-Cowling Cowling (1966). technique

Ball-milled cellulose. This cellulose was ob- The technique employed by Rautela and tained from Dr. H.O.W. Eggins and Bernard Cowling (1966) for testing the cellulolytic King at the Biodeterioration Information activity of fungi was adopted. Vertical agar Centre, University of Aston in Birmingham. columns (approx. height 40 mm), containing the various substrates were prepared in test HCI-cellulose. Cellulose powder (Whatman tubes (18 mm diam.). The two media descri- CF 11) treated with concentrated hydro- bed in a previous study (Nilsson 1973) were also used here. The media had the following Walseth cellulose 0.25 R-C compositons: Walseth cellulose 0.125 R-C Walseth cellulose 0.25 B-VII R-C medium (after Rautela and Cowling Walseth cellulose 0.125 B-VII 1966) Ball-milled cellulose 0.2 B-VII NHqH2P04 2.0 g, KH2P04 0.6 g, K2HP04 Ball-milled cellulose 0.2 A 0.4 g, MgS04' 7 H20 0.89 g, thiamine HC1 Ball-milled cellulose 0.2 B 100 pg, yeast extract 0.5 g, adenine 4.0 mg, HC 1-cellulose 0.125 B-VII adenosine 8.0 mg, agar 17 g and distilled Xylan 0.125 B-VII water to make 1 liter. Glucomannan 0.1 B-VII Starch 0.2 B-VII B-VII medium (slight modification of Brav- ery's (1968) medium VII): The test tubes were fitted with cotton plugs (NH4)2S04 0.543 g, KH2P04 1.0 g, KC1 and sterilized by autoclaving. The tubes were 0.5 g, MgS04 7 H20 0.2 g, Ca C12 0.1 g, agitated by hand during the cooling of the thiamine HC 1 1 mg, agar 15 g and 1000 ml agar in order to keep the carbohydrates of deionized water. uniformly suspended. The tubes were left in upright position when the agar solidified in In addition to these media, two further order to obtain vertical columns. Opaque media were employed in test tubes with agar columns were obtained since the sub- cellulose. These media had the following strates used were insoluble, or only partly composition : soluble, in water.

Medium A Most of the test tubes were inoculated with (NH4)2S04 2.5 g, KH2PO4 1.0 g, KC1 0.1 small pieces (approx. 2x2 mm) of mycelium g, MgS04' 7 H20 0.5 g, Ca C12 0.1 g, and agar taken from actively growing cultu- FeS04 ' 7 H20 10 mg, CuS04 ' 5 H20 10 res on malt extract (2.5 percent) agar. The mg, yeast extract 0.5 g, agar 15 g and 1000 test tubes with Walseth cellulose and ball- ml of deionized water. milled cellulose in B-VII medium were, however, inoculated with spores or aerial Medium B mycelium in order to avoid the addition of The same as medium A but 3.0 g KNO3 malt extract from the agar plates. Each instead of (NH4)2S04. fungus was inoculated in two replicate tubes. All test tubes were incubated, standing The media R-C, A and B contain yeast perpendicular, at the ambient room tempera- extract which might be used as a carbon ture (23 - 25'~). Some of the fungi were source by certain fungi. Medium B-VII also incubated at 15 and 30'~. The cotton contains no yeast extract. The media A and plugs were covered with an aluminium foil B have a higher nitrogen content than media to prevent the agar from drying out. R-C and B-VII. If the amount of nitrogen is calculated from the nitrogen containing The depth of clearing was measured every salts, medium A contains 530 mg, medium B week for up to twelve weeks. If clearing had 41 5 mg, medium R-C 243 mg and medium occurred after three or six weeks, no further B-VII 115 mg N per liter. measurements were taken. The depth of clearing was measured in millimeters from The different carbohydrates to be tested the top of the agar to the front of the clear were added to the four media in the follow- zone. ing combinations:

Carbohydrate Concentration Medium 2.4 Cultivation on cellulose agar plates and percent (Wlv) assay of the cellulase produced. AviceI 0.125 R-C Avicel 0.125 B-VII Cellulose agar plates were prepared in 90 mm plastic Petri dishes with the following et al. (1967) was used to assay the cellulase two media: produced in the cellulose agar media. This method employs the transfer of mycelium- F6A agar plugs from the cellulose agar plates Avicel 10 g, asparagine 1.0 g, NH4N03 1.0 where the fungi have been growing. The agar g, KH2P04 1.0 g, MgS04 7 H20 0.5 g, plugs are cut with a cork-borer and transfer- FeS04 ' 7 Hz0 10 mg, .ZnSOq 7 H20 10 red to a hole in a test agar plate which mg, glucose 2.5 g, yeast extract 0.5 g, agar contains suspended cellulose. The cellulase 15 g and 1000 ml of deionized water. present in the transferred plug will diffuse out into the surrounding agar and produce This is the same cellulose agar medium a clear zone. Savory et al. used an addition which was used in a previous study (Nilsson of sodium azide (0.005 mole/l) to the test 1973) on the wood attack produced by agar in order to prevent growth of the fungi. micro fungi. Two types of test agar plates were used in B- VII cellulose agar this study. One was prepared with Rautela- 10 grams of Avicel was added to 1 liter of the Cowling medium which was poured on medium B-VII described above. plates instead of into the test tubes. The concentration of Walseth cellulose was 0.25 The media were autoclaved and the plates percent. The other plates contained the same were poured when the agar had cooled to medium and amount of Walseth cellulose, but about 45O~.The media were agitated when sodium azide (0.3 g/l) was also added. pouring in order to suspend the cellulose particles in the agar. Mycelium-agar plugs were cut with a cork- borer (10 mm diam). from the plates with The agar plates with medium F6A were F6A and B-VII cellulose agar on which the inoculated with small pieces of mycelium fungi had grown for three, six or fifteen and agar taken from actively growing cultu- weeks. Two plugs were taken from each res on malt extract (2.5 percent) agar. The plate; one plug was taken close to the centre agar plates with B-VII medium were inocu- of the colony while the other was taken lated with spores or pieces of aerial mycelia. immediately behind the margin of the colo- Three agar plates were inoculated with each ny. The agar plugs were transferred to holes fungus and each medium. For growth com- cut with the same cork-borer in the test agar parisons each fungus was also inoculated on plates. The plugs were inserted in the holes three plates with malt extract (2.5 percent) with the fungal growth uppermost. Approxi- agar. mately ten agar plugs were placed in each test plate. The agar plates were placed in perforated plastic bags and incubated at the ambient The test plates without sodkm azide were room temperature for periods of three and incubated for two days at 40 C. This tempe- six weeks. Some of the fungi growing on rature was employed in order to prevent B-VII cellulose agar were incubated for fungal growth without the use of any poiso- fifteen weeks. nous additives. It is also known that cellula- ses generally have a higher activity at 40' After incubation a visual estimation of the than at 23-25', which was the room tempe- growth on the cellulose agar plates was rature here. The test plates with sodium made. The growth was compared with the azide were incubated for two days at room growth on malt extract agar. temperature.

The cellulose agar plates were also examined After incubation the test plates were exami- for any visible clearing under or around the ned for clearing around the agar plugs. No fungal colonies. measurements of the width of the clear zones were made, only the presence or The agar-diffusion test described by Savory absence of clearing being noted. 2.5 Assay of cellulase and xylanase present to grow in the wood sections since the test in wood blocks attacked by the test agar was poisoned with sodium azide. The organisms. enzymes present in the wood at the time of sectioning will diffuse out, if diffusible, in Decay tests were carried out with birch sap the agar and produce clear zones in the wood (Betula verrucosa) on cellulose and substrate (see Fig. 3). Henningsson et al. malt extract agar slopes in test tubes accor- (1972) used this method to demonstrate the ding to the methods described in a previous presence of cellulase and xylanase in birch paper (Nilsson 1973). Each fungus was wood chips attacked by a white rot fungus. cultivated on the medium which had previ- This method is extremely sensitive, at least ously been found to support the heaviest for the detection of cellulase, as is evident attack. from dilution tests with a cellulase-contain- ing solution (see section 2.6). The test agar After varying periods of time, usually after plates were incubated for up to five days at approximately 20,40 or 60 days, the attack- 40'~. After incubation the test plates were ed wood blocks were removed from the examined for clearing under or around the test tubes and immediately sectioned with a wood sections. razor blade. Both transverse and longitudinal sections were cut. The sections were 0.5 to 1 mm thick and the sides were 3 to 5 mm. 2.6 Cultivation in liquid media. Weight los- ses of cellulosic substrates and assays of The sections were placed on two types of cellulase and xylanase. test plates immediately after cutting. The test plates were prepared according to The two following liquid media were used: Stranks and Bieniada (1971) in 90 mm plastic Petri dishes. Medium EP (NH4)2 SO4 0.5 g, DL-asparagine 0.5 g, The test plates were prepared as a twin agar KH2P04 1.0 g, KC1 0.5 g, MgS04 7 H20 where the bottom layer contained agar (1 0.2 g, CaCI2 0.1 g, yeast extract 0.5 g and percent), sodium azide (0.005 percent) and deionized water 1000 ml. 30 ml of 0.2 M sodium acetate buffer pH 5.5 per 100 ml. The top layer contained the After autoclaving together with 100 mg same ingredients but the agar concentration cellulose N the pH was 5.4. was reduced to 0.5 percent. The substrate to be tested, in this case cellulose or xylan, was Medium EP contained the same ingredients added to the top layer. When the agar had as the medium described by Eggins and Pugh solidified, the substrate formed a thin mono- (1962), except for the agar and ball-milled layer between the two agar layers. The top cellulose present in the Eggins and Pugh layer in the test plates used here for assay of medium. cellulase activity contained 0.25 percent Walseth cellulose instead of the recommen- Medium B- VII-L ded 2 percent. The top layer in the test As medium B-VII described previously, but plates for the assay of xylanase activity since it was a liquid medium, no agar was contained 2 percent larch xylan. added. In addition to thiamine, medium B-VII-L also contained 0.2 mg biotin and Stranks and Bieniada suggested the use of 0.2 mg pyridoxin per 1000 ml. these plates for detection of cellulase and hemicellulases in culture filtrates. The for- The pH was 4.6 after autoclaving together mation of clear zones around small droplets with 100 mg cellulose N. of culture filtrate added to the test plates indicate cellulase or hemicellulase activity. The fungi were cultured in 100 ml Erlen- In the present study, sections from attacked meyer flasks. In most experiments 20 ml wood were used instead of culture filtrate. medium was added to each flask together Tests showed that the fungi did not continue with 100 mg of the cellulose substrate (weighed air-dry). The flasks were sterilized EP medium and the cellulose substrates were by autoclaving. Each flask was inoculated used as controls. with 2 ml of a spore or mycelial suspension. Three or four replicate flasks were used in After incubation the mycelium and remain- each test. Both shake and stationary cultures ing cellulose were filtered off in pre-weigh- were used. The shake cultures were placed in ed glass crucibles and washed several times a rotary shaker at 125 revolutions per with distilled watoer. The crucibles were dried minute. All cultures were incubated at am- overnight at 105 C and weighed. The weight bient room temperature. loss of the cellulose substrates was calculated as the difference between the added amount of cellulose (100 mg) and the dry weight of Growth on medium EP and B-VII-L with the remaining cellulose plus mycelium. The glucose as carbon source weight losses reported were not corrected for the weight of mycelium, nor were they All fungi which had failed to produce corrected by the values obtained from the clearing of cellulose in the tests with the non-inoculated control flasks. Rautela-Cowling technique and some of the fungi which had produced clearing only on The pH of the culture filtrates was measured B-VII medium were tested on the liquid and is reported as the final pH. The culture media EP and B-VII-L in order to see if filtrates were also used for assays of cellulase these media could support growth of the and xylanase. The test agar plates recom- fungi. For this purpose glucose, 20 g/liter, mended by Stranks and Bieniada (19711, but was added to 1000 ml of each of the media. slightly modified as described in the pre- As a control, some of the fungi were grown ceeding section (2.5), were used for the on medium EP without glucose. Only statio- assays. 80 pl of culture filtrate was added to nary cultures were employed. The flasks a test plate with Walseth cellulose and 80 pl were harvested after 14 or 15, 21 and 28 of the same filtrate was added to a test plate days, except for flasks containing medium with larch xylan. Up to twenty droplets B-VII-L which were harvested after 21 could be put on the same test plate if the days. The mycelia were filtered off in glass enzyme activity was low. If enzyme activity crucibles and were dried and weighed to was high, a maximum of six droplets could ascertain the dry weights of mycelium. pH be added to each plate. was measured in the culture filtrates. The test plates were incubated at 40'~for Determination of weight losses of cellulose two days and they were then examined for N, Avicel and cotton produced by the test clear zones. This incubation time was chosen fungi. Assay of cellulase and xylanase in since some of the filtrates showed such low culture filtrates. activity that no clearing was perceptible after only one day. In the beginning of the 20 ml of medium was added to each flask experiments, some plates were kept for up together with 100 mg of the cellulose to seven days but no case was found where substrate. All of the fungi were tested on clearing was perceptible first after three or medium EP with cellulose N as substrate, more days. Some measurements were made some of them were also tested on Avicel and of the width of the clear zones, but since cotton. All of the species which failed to exact measurements were difficult due to show activity on the EP medium were tested diffuse clearing and diffuse zone fronts, only on B-VII-L medium with cellulose N as the presence or absence of activity is report- substrate. Some of these species were also ed in the results presented. tested on B-VII-L medium with Avicel or cotton as substrate. A number of the species The sensitivity of the test method was which were active on EP medium were also studied here with a solution of cellulase cultivated on B-VII-L medium for compar- from Sporotrichum pu2verulentum Novobra- ison. Both shake and stationary cultures nova obtained from Dr. B. Pettersson at the were employed. Non-inoculated flasks with Swedish Forest Products Research Laborato- ry. This cellulase solution released 1.6 mg 500 mg of substrates (weighed air-dry) was reducing sugar per ml of an Avicel suspen- added to each 100 ml Erlenmeyer flask sion (1 %) after 4 hours at 30'~. Several together with 5 ml of medium EP. The flasks dilutions were prepared of the cellulase were inoculated and incubated as stationary solution with distilled water. 80 pl of each cultures. Weight loss determinations, pH dilution was added to a test plate containing measurements and assays of cellulase and Walseth cellulose. Perceptible clearing was xylanase were carried out as described abo- still obtained at a 400-fold dilution. This ve. Non-inoculated flasks were used as cont- shows that the method is very sensitive for a rols. detection of cellulase. Some microscopic studies of the exposed fibres were carried out after weighing. Small Growth in liquid cultures with birch wood amounts of the fibres were spread on a glass meal. Assay of cellulase and xylanase. slide, mounted in a glycerol-water mixture (1 :1) and viewed under a light microscope A few of the fungi were tested in medium using polarized light. EP and medium B-VII-L with birch wood meal as the substrate. 100 mg wood meal (dried overnight at 105'~) was added to Effect of various amounts of glucose on the each flask plus 20 ml of the medium. Both production of cellulase and xylanase shake and stationary cultures were employ- ed. Weight loss determinations and assays of Bispora betulina, Ceratocystis albida, Cla- cellulase and xylanase were performed as dorrhinum sp. A, Phialocephala sp. A, Xylo- described above. gone sphaerospora and Fungus A were grown in stationary cultures in flasks con- taining 20 ml of medium EP to which Determination of weight losses of cotton varying amounts of glucose had been added. and jute fibres Approximately 100 mg of cellulose N added to each flask served as cellulose substrate. Five of the test fungi, Bispora betulina, The flasks were harvested after the incuba- Ceratocystis albida, sp. A, War- tion times given in Table 9. The mycelium domyces inflatus and Xylogone sphaero- and cellulose were filtered off and discarded. spora were grown in medium EP on cotton pH was measured in the culture filtrates and and jute fibres in order to compare the assays of cellulase and xylanase in the degradation of non-lignified and lignified filtrates were made on test plates as describ- fibres. ed above. 3 Results

3.1 Type of attack produced in birch wood the table since clearing was similar to that occurring on medium B-VII. The type of attack produced by the fungi in birch wood is shown in Table 2. These data The depth of clearing varied considerably are taken from a previous paper (Nilsson among individual species. It was also depen- 1973), except for the four species mention- dent on time of incubation, type of medium ed in Materials and methods. Their decay and type of substrate. Fig. 1 shows the patterns were studied and it was found that amount of clearing produced by Xylogone Ceratocystis albida and Ceratocystis steno- sphaerospora, Cordana pauciseptata, Bispora ceras only produced soft rot cavities and no betulina and Cladosporium resinae in test erosion in birch wood. Cladosporium sp. A tubes with B-VII medium containing 0.2 and Fungus D produced a weak erosion of percent ball-milled cellulose. the cell walls but no cavities. The greatest clearing zones in the agar columns with cellulose as the substrate were 3.2 Clearing produced in agar columns with obtained with Walseth cellulose. Smaller cellulose, xylan, glucomannan and clearing zones were obtained on ball-milled starch (Rautela-Cowling technique). cellulose and these zones tended to be more diffuse. Only a few of the fungi were able to The average depth of clearing in two repli- produce any measurable clearing on Avicel cate tubes after 21 and 42 days on the and HC 1-treated cellulose. various substrates is given in Table 2, Walseth cellulose was used in 0.25 percent concen- If clearing of Walseth cellulose and Avicel on tration. The concentrations of the other the two media, R-C and B-VII, is compar- substrates are given in Materials and me- ed, it is obvious that greater clearing zones thods. The depth of clearing of ball-milled were always obtained on medium B-VII. All cellulose on media A and B is not shown in of the species which produced clearing of

Figure 1. Amount of clearing after 42 days in test tubes containing 0.2 percent ball-milled cellu- lose in B-VII medium. A. Xylogone sphaerospora, B. Cordana pauciseptata, C. Bi- spora betulina, and D. Clado- sporium resinae. Distinct clearing is evident in tubes A and B. No clearing occurred in tubes C and D although a fair amount of growth was produced by B. betulina and C. resinae, which can be seen as a dark colouration in the agar columns. Walseth cellulose on R-C medium also cellulose. But Acremonium atro-griseum, produced clearing of Walseth cellulose and Humicola alopallonella, Petriellidium boydii ball-milled cellulose on B-VII medium. The and Phialocephala dimorphospora which also difference in the amount of clearing on these produced clearing of the cellulose, have been media was, however, considerable for some found to be unable to cause any detectable of the fungi. Acremonium atro-griseum, Cla- erosion-type attack in birch wood. These dorrhinum sp. A, Dictyosporium elegans, species only form soft rot cavities in the and Wardomyces infla- birch wood (Type 1 attack). Similar results tus, which all produced less than a 1 mm were already obtained in a previous study clear zone of Walseth cellulose on R-C (Nilsson 1973). medium, produced clear zones of 9 to 13 mm on B-VII medium with the same The following sixteen species failed to pro- substrate. duce clearing on any of the cellulose agars tested: Bispora betulina, Catenularia heimii, pannorum was the only Ceratocystis albida, Ceratocystis olivacea, species which produced clearing of Avicel on Ceratocystis stenoceras, Cladosporium resi- R-C medium. When B-VII medium was nae, Gonatobotrys sp. A, Graphium sp. A, used, ten species produced some clearing of Mollisia sp. A, Phialocephala sp. A, Phialo- Avicel, and twelve species produced clearing cephala sp. C, , Phialo- of the HC 1 cellulose. The amount of clearing phora sp. A, Rhinocladiella sp. A, Fungus A was, however, considerably less than that and Fungus B. produced on Walseth cellulose. These fungi were incubated for up to 12 Cladosporium sp. A, Humicola alopallonella, weeks on Walseth cellulose with R-C and Phialocephala dim orphospora, Scy talidium B-VII medium, but even after this time no sp. B and Fungus D all failed to produce any clearing had occurred. Tests on cellulose agar clearing of cellulose on R-C medium, even with a lower concentration of Walseth cellu- if the concentration of Walseth cellulose was lose, 0.125 percent instead of 0.25 percent, reduced to 0.125 percent. Clearing was, also gave negative results. The sixteen spe- however, obtained on B-VII medium with cies were also tested on B-VII medium with 0.25 percent of Walseth cellulose. Phialoce- 0.125 percent Walseth cellulose at 15 and phala dimorphospora failed to produce clear- 30'~ for up to six weeks, but still no ing of ball-milled cellulose on B-VII med- clearing was obtained. ium, while the remaining four species pro- duced clearing. Of these five fungi, only H. All of these fungi produced at least some alopa1lonella was able to produce a slight growth on the cellulose agars. As can be seen clearing of HC 1 cellulose. in Fig. 1, some of them produced a fair amount of growth without being able to All of the species which produced clearing of form any clearing zones. To ensure that Walseth cellulose on R-C medium also clearing was not obscured by the mycelia produced clearing of ball-milled cellulose on which penetrated into the agar, microscopic media A and B. The depth of clearing was studies of the top layers of the agar columns very similar between medium A and medium were carried out as described in a previous B. Almost equally large zones were formed paper (Nilsson 1973). However, no evidence in ball-milled cellulose on medium B-VII as of degradation of the cellulose was noted for on media A and B. Humicola alopallonella, any of the species mentioned above. Phialocephala dimorphospora, Scytalidium sp. B and Fungus D failed, however, to Only three of the sixteen species, viz. produce any clearing on media A and B. Ceratocystis olivacea, Cladosporium resinae and Phialophora verrucosa, are unable to It is evident from Table 2 that all of the produce other attack in birch wood than species which produced an erosion-type at- small bore holes through the cell walls. The tack (Type 2) in the birch wood also remaining thirteen species all produce soft produced at least some clearing of the rot cavities in birch wood (see Table 2). All species which produced clearing of cellu- rium resinae, Graphium sp. A, Mollisia sp. A lose, except Humicola alopallonella and and Phialocephala sp. A produced clearing of Phialocephala dimorphospora, also produced xylan, but not of glucomannan. The remain- clearing of xylan and glucomannan. H. alo- ing eight species: Catenularia heimii, Cerato- pallonella produced no clearing of glucoman- cystis albida, Ceratocystis olivacea, Cerato- nan and P. dimorphospora produced no cystisstenoceras, Gonatobotrys sp. A, Phialo- clearing of xylan. cephala sp. C, Phialophora verrucosa and Rhinocladiella sp. A failed to produce clear- Of the sixteen species which failed to ing of both xylan and glucomannan. produce clearing of any of the cellulose substrates, the following formed clear zones Phialophora verrucosa was the only species in both xylan and glucomannan agar: Phialo- which failed to produce clearing of starch. phora sp. A, Fungus A and Fungus B. Five All of the other species already produced of the species, Bispora betulina, Cladospo- appreciable clearing after 21 days.

Figure 2. Comparison of growth of Petriellidium boydii(A),Phia- lophora sp. A (B) and Rhinocladzella sp. A (C) on malt extract (2.5 %) agar (right) and F6A cellulose agar (left). 3.3 Growth and cellulase production on It was difficult, due to obscuring mycelia, to cellulose agar plates observe any clearing of the cellulose agar on which the fungi grew. However, the follow- All results from these experiments are shown ing species produced visible clearing in F6A in Table 3. cellulose agar: Cladorrhinurn sp. A, Conio- thyrium fuckelii var. sporulosum, Humicola There were great variations in the amount of grisea, Petriellidium boydii, Phialophora growth produced on the three types of agar hoffmannii, Scytalidium lignicola and Xylo- media employed. Growth was generally best gone sphaerospora. Cladosporium sp. A, on malt extract agar, followed by F6A Scytalidium sp. B and Fungus D produced cellulose agar and less on B-VII cellulose visible clearing on B-VII but not on F6A agar. Fungi such as Cladorrhinurn sp. A, medium. Coniothyrium fuckelii var. sporulosum, Hu- micola grisea, Petriellidum boydii, Phialo- No differences in cellulolytic activity were phora fastigiata, Phialophora hoffmannii, shown if the test plates were incubated at Scytalidium lignicola, Wardomyces inflatus 40'~or if the test agar was poisoned with and Xylogone sphaerospora produced equal sodium azide. Thus, separate results with the or better growth on F6A cellulose agar when two types of test plates are not shown in compared with malt extract agar. All of Table 3. Cladorrhinum sp. A and Petrielli- these species also produced clearing of cellu- dium boydii produced zome growth on the lose with the Rautela-Cowling technique (see plates incubated at 40 C but this did not Table 2). Although Cordana pauciseptata, obscure the clearing. Rhinocladiella anceps and Scy talidium sp. B also produced clearing of cellulose with the All of the species which had produced same technique their growth was sparse on clearing of cellulose with the Rautela-Cow- F6A cellulose agar. The remainder of the ling technique on R-C medium, except for tested species showed sparse or moderate Rhinocladiella anceps, were found to produ- growth on F6A cellulose agar. Fig. 2 shows ce cellulase when growing on F6A cellulose the growth of three species on F6A cellulose agar. But Humicola alopallonella, Scytali- agar as compared with growth on malt dium sp. B and Fungus D only produced extract agar. cellulase when they grew on B-VII cellulose agar. Irrespective of media, no celluloytic All species, except Scytalidium sp. B, pro- activity was found for Phialocephala dimor- duced much less growth on B-VII cellulose phospora or Rhinocladiella anceps, nor for agar as compared with malt extract agar and any of the sixteen species which had failed in most cases also when compared with F6A to produce clearing of cellulose with the cellulose agar. Humicola alopallonella, Scyta- Rautela-Cowling technique (see section 3.2), lidium sp. B and Fungus D were the only even if the incubation time was extended to species which produced equal or increased fifteen weeks. growth on B-VII than on F6A cellulose agar. Scytalidium sp. B grew almost equally well on B-VII cellulose agar as on malt 3.4 Presence of cellulase and xylanase in extract agar. birch wood attacked by the test orga- The truly non-celluloytic fungi Ceratocystis nisms olivacea, Cladosporium resinae and Phialo- phora verrucosa produced equal or better Thirty of the test fungi were used in growth on both of the two cellulose agar experiments to demonstrate the presence of media than several of the species which were cellulase and xylanase in attacked birch able to produce soft rot in birch wood but wood. No cellulolytic activity had been which had failed to produce clearing in found for fourteen of these species when cellulose agar columns. Such species were using the two methods described previously, Catenularia heimii, Ceratocystis albida, C. and six of the species had failed to produce stenoceras, Gonatobotrys sp. A, Phialoce- clearing of xylan with the Rautela-Cowling phala sp. C and Rhinocladiella sp. A. technique. Figure 3. Clearing of Walseth cellulose (left) and xylan (right) a- round sections cut from birch wood blocks attacked by Xylogone sphaerospora for 27 days. Sodium azide is incorporated in the test agar plates to prevent growth of the fungus.

The results areillustrated in Table 4. Clearing also shown cellulolytic activities in the pre- of the Walseth cellulose in the test plates was vious tests. However, no clearing of the obtained around sections cut from birch cellulose was found around sections cut wood blocks attacked by sixteen of the from wood attacked by Dictyosporium ele- thirty fungi. Among these sixteen species guns, Humicola alopallonella, Petriellidium were four species, Bispora betulina. Phialoce- boydii and Wardomyces inflatus, although phala sp. A, Phialophora sp. A and Fungus micioscopic examination of the wood blocks A, which had not shown any cellulolytic from which the sections had been cut, activity in the two previous tests. Fig. 3 showed that substantial degradation had shows clearing of Walseth cellulose and occurred. xylan around sections cut from birch wood attacked by Xylogone sphaerospora. The presence of xylanase in wood was No clearing of cellulose was obtained around closely correlated with the presence of sections cut from wood blocks attacked by cellulase. Except for three of the fungi. viz. Catenularia heimii, Ceratocystis albida, Cera- Bispora betulina, Ceratocystis stenoceras and tocystis stenoceras, Cladosporium resinae, Pseudeurotium zonatum, xylanase was al- Gonatobotrys sp. A, Graphium sp. A, Molli- ways found in wood blocks together with sia sp. A, Phialophora verrucosa, Rhinocla- cellulase. Cellulase, though not xylanase, diella sp. A and Fungus B. All of these activity was detected in wood blocks attack- species had also failed to show cellulolytic ed by Bispora betulina and Pseudeurotium activity in the two previous tests. Microsco- zonatum whereas xylanase but not cellulase pic examination of the attacked wood was detected in wood blocks attacked by blocks from which the sections had been cut Ceratocystis stenoceras. showed that all of these species, except Cladosporium resinae, Mollisia sp. A and Xylanase activity could not be detected Phialophora verrucosa, had caused conside- around sections cut from wood attacked by rable degradation of the wood in the form of nine of the twenty-four species, which pre- soft rot cavities. Cladosporium resinae and viously had been found to produce clearing Phialophora verrucosa had only produced of xylan with the Rautela-Cowling techni- bore holes through the wood cell walls while que. Of all of the species which had failed to Mollisia sp. A had produced a few cavities. produce clearing of xylan with the same technique, Ceratocystis stenoceras was the Cellulase was demonstrated in the birch only species for which xylanase could be wood for twelve of the species, which had demonstrated in attacked wood. 3.5 Growth on medium EP and B-VII-L in Rautela-Cowling technique. Most of the liquid cultures with glucose as the sour- species which had produced clearing of ce of carbon cellulose were also able to cause weight losses of cellulose and cellulase and xylanase The results illustrated in Table 5 show that could be found in the culture filtrates. all of the tested fungi could grow on the two media, EP and B-VII-L, with glucose as the The weight losses were generally higher on source of carbon. No growth or very slight medium EP than on medium B-VII-L. growth occurred on medium EP without Exceptions were found for Acremonium glucose. a t ro-griseum and Cordana pauciseptata which produced higher weight losses of Phialocephala sp. C and Rhinocladiella sp. A cotton on medium B-VII-L than on EP showed slightly better growth on B-VII-L medium. Their weight losses of Avicel and than on EP medium. All of the other species cellulose N were, however, higher on EP produced equal or, in some cases, consider- than on B-VII-L medium. Rhinocladiella ably more growth on EP medium. anceps and Scytalidium sp. B were also exceptional in causing higher weight losses All of the species lowered the pH of both of of cellulose N on B-VII-L medium than on the media during growth on glucose. A rise EP medium. The last two fungi produced no in pH was noted in the flasks with medium weight losses of cellulose N on EP medium EP without glucose. after 42 days whereas the weight losses on B-VII-L medium were 26.8 and 46.0 per- Differences in growth rates between the test cent respectively after 2 1 days. species grown on EP medium can be noted in the table. Some species had evidently On medium B-VII-L only very few of the reached their maximal mycelial weights al- fungi produced cellulase and xylanase which ready after 14 days, while others still show- could be detected in the culture filtrates. ed an increase in mycelial weight up to 28 This also applies to species which caused days. significant weight losses on B-VII-L me- dium.

3.6 Degradation of cellulose N, Avicel and Differences were noted between shake and cotton and the production of cellulase stationary cultures. Coniothyrium fuckelii and xylanase on these substrates in var. sporulosum, Pseudeurotium zonatum, liquid cultures Scytalidium lignicola and Fungus D caused higher weight losses on medium EP in shake The results with medium EP are shown in cultures than on the same medium in statio- Table 6 and the results with medium B-VII nary cultures, while the reverse was found -L are illustrated in Table 7. The weight for Dictyosporium elegans, Humicola alo- losses reported represent the average of three pallonella, Phialophora fastigiata and Fungus or four flasks. A (see Table 6). Especially great differences were noted for Phialophora fastigiata grow- The non-inoculated controls with EP as the ing on cotton in EP medium. After 42 days medium showed weight losses of the cellu- the weight loss of cotton in stationary lose substrates which ranged from 0.8 to 1.0 cultures was 38 percent while only 1 percent percent in shake cultures and from 2.1 was lost in shake cultures. Fungus A behaved percent to 5.1 percent in stationary cultures. similarily on cellulose N on EP medium. The Since great errors are involved in the proce- weight loss was nearly 46 percent after 42 dure of determining weight losses of the days in stationary cultures while no weight cellulose substrates, only weight losses above losses occurred in shake cultures. 10 percent are regarded as significant. If the weight losses obtained in medium EP The results show a pattern similar to that of for the different are compared, it the results of the clearing tests using the appears that cellulose N in most cases was more rapidly degraded than Avicel, while If weight loss is used as a criterion for cotton was the most resistant substrate. But cellulolytic activity, the following fungi, individual variations were found, Chryso- which caused more than 10 percent weight sporium pannorum, for instance, caused a loss, may be considered cellulolytic: Acre- higher weight loss of Avicel than of cellulose monium atro-griseum, Chrysosporium pan- N. Some of the fungi showed considerable norum, Cladorrhinum sp. A, Coniothyrium variations in their ability to degrade the fuckelii var. sporulosum, Cordana paucisep- different cellulose substrates. Acremonium tata, Dicty osporium elegans, Humicola alo- atro-griseum caused weight losses of 3 5.0 pallonella, Humicola grisea, Petriellidium and 48.4 percent of Avicel and cellulose N boydii, Phialophora fastigiata, Phialophora respectively, while the weight loss of cotton hoffmannii, Pseudeurotium zonatum, Rhino- was only 4.1 percent. When medium B-VII cladiella anceps (only on medium B-VII -L was used, the fungus produced during -L), Scytalidium lignicola, Scytalidium sp. the same time a weight loss of 33.2 percent B (only on medium B-VII-L), Wardomyces of cotton while Avicel and cellulose N lost inflatus, Xylogone sphaerospora, Fungus A only 15.2 and 13.5 percent respectively. and Fungus D.

Dictyosporium elegans and Fungus A caused Both cellulase and xylanase were detected in weight losses only in cellulose N. Avicel and culture filtrates from all the species mention- cotton were not attacked. Phialophora fasti- ed above, except for the culture filtrates giata did not degrade Avicel but caused high from Humicola alopallonella and Scytali- weight losses of both cotton and cellulose N. dium sp. B in which no xylanase was detected. Among the remainder of the tested Among the species which were tested on species Cladosporium sp. A was found to medium EP, the following produced cellu- produce cellulase and Mollisia sp. A and lase and xylanase that could be detected Phialophora sp. A were found to produce in the culture filtrates, both in shake and xylanase. None of these enzymes was detec- stationary cultures: Coniothyrium fuckelii ted in culture filtrates from the rest of the var, sporulosum, Phialophora fastigiata, Scy- fungi. talidium lignicola and Fungus D. Dictyo- sporium elegans and Pseudeurotium zonatum The pH after cultivation was higher than the produced both cellulase and xylanase in pH of the non-inoculated controls on me- stationary cultures but in culture filtrates dium EP. The highest pH values were found from shake cultures no xylanase could be in culture filtrates from flasks where no detected. The reverse was found for Petrielli- cellulose degradation had occurred. On me- dium boydii. Humicola alopallonella and dium B-VII-L, the pH was the same or Fungus A did not produce any of the two lower than the pH of the medium after enzymes in shake cultures, both produced autoclaving. Especially low pHs were mea- cellulase in stationary cultures and Fungus A sured in culture filtrates from flasks with also produced xylanase. Mollisia sp. A pro- medium B-VII-L where significant degrad- duced xylanase but not cellulase in shake ation of the cellulose had occurred. cultures. None of the enzymes were found in stationary cultures. 3.7 Degradation of birch wood meal and On medium B-VII-L comparisons can only production of cellulase and xylanase on be made for Phialophora sp. A and Fungus this substrate in liquid cultures D. Cellulase and xylanase were detected in stationary cultures of Fungus D, while no Chrysosporium pannorum, Bispora betulina, xylanase was found in shake cultures. Phialo- Phialophora sp. A and Phialocephala sp. A phora sp. A showed xylanase activity on were grown in liquid cultures on birch wood Avicel after 42 days. No cellulase activity meal as described in Materials and methods was found on any of the substrates in (section 2.6). The incubation was extended stationary cultures. None of the enzymes to 42 days but even after this time no were found in shake cultures. significant weight losses were obtained, nor was cellulase and xylanase detected in any of xylanase were detected in filtrates from jute the culture filtrates. fibre cultures, whereas only cellulase was detected in the cultures with cotton. Only an erosion type of attack was seen under the microscope in both types of fibres. The pH of the culture filtrates was lower, especially 3.8 Degradation of cotton and jute fibres the pH of filtrates from cultures with cot- and production of cellulase and xylana- ton, than the pH of the controls. se on these substrates in liquid cultures

Table 8 shows the results obtained from liquid cultures with cotton and jute fibres as substrates. 3.9 Effect of various amounts of glucose added to medium EP on the production Bispora betulina and Phialophora sp. A did of cellulase and xylanase. not cause any significant weight losses of cotton and jute fibres, nor could cellulase Table 9 shows the influence of the addition and xylanase be detected in the culture of various amounts of glucose to the EP filtrates. No degradation of any of the two medium on the production of cellulase and fibres was observed in the microscope. xy lanase.

Judging from the small and insignificant The addition of up to 0.1 percent glucose as weight losses, no attack was caused by "start-glucose" did not stimulate Bispora Ceratocystis albida on cotton fibres. Neither betulina, Ceratocystis albida or Phialoce- could any degradation of the cotton fibres phala sp. A to produce cellulase or xylanase. be seen in the microscope. The pH of the Table 9 shows that no cellulase or xylanase culture filtrates was considerably higher than could be detected in any of the culture that of the non-inoculated controls. Signifi- filtrates. The pH was higher than that of the cant weight loss was, however, obtained in autoclaved EP medium before inoculation. the jute fibres, 30.8 percent after 42 days. Microscopic examination of the jute fibres It can be noted in Table 9 that increasing revealed numerous soft rot cavities. Cellulase amounts of glucose retarded the production and xylanase could not, however be detected of cellulase and xylanase by Cladorrhinum in the culture filtrates. The pH of the sp. A, Xylogone sphaerospora and Fungus A. filtrates was lower than that of the controls. The addition of glucose had a greater in- fluence on the production of xylanase than Wardomyces inflatus was capable of attack- on that of cellulase. 0.25 percent glucose ing both cotton and jute. After 42 days the prevented the formation of detectable weight losses were 12.9 and 19.8 percent amounts of xylanase by Cladorrhinum sp. A respectively. Microscopical examination re- and Xylogone sphaerospora. The latter spe- vealed erosion in the cotton fibres and cies produced detectable cellulase at 0.5 erosion and soft rot cavities in the jute percent glucose, whereas cellulase could not fibres. Both cellulase and xylanase were be found for Cladorrhinum sp. A at this detected in the culture filtrates from the glucose concentration. flasks with cotton after 42 days, though neither of these enzymes appeared in the Fungus A produced cellulase after seven jute cultures. The pH of the culture filtrates days in cultures with 0.005 and 0.01 percent from both of the substrates was higher than glucose, but none in cultures with 0.025 the pH in the control flasks. percent. However, after 14 days cellulase was found in cultures with 0.1 percent Xylogone sphaerospora was the only species glucose, which was the highest concentration which produced a higher weight loss of tested with this fungus. Xylanase was also cotton than of jute, 59.5 and 27.2 percent found after 14 days in cultures with 0.1 respectively, after 42 days. Cellulase and percent glucose. The pH of the culture filtrates from Clador- method. Tests for mannanase and amylase rhinum sp. A, Xylogone sphaerospora and were carried out only with the Rautela- Fungus A were in most cases lower than the Cowling technique so no comparisons can be start pH, especially if increasing amounts of made with the other methods. glucose had been added. All species except Phialophora verrucosa produced amylase. The following twenty species were found to also produce all of the three other enzymes: Acremonium atro- 3.10 Comparison of the results obtained by griseum, Chrysosporium pannorum, Clador- the different methods rhinum sp. A, Cladosporium sp. A, Coniothy- rium fuckelii var. sporulosum, Cordana pau- Table 10 shows which of the enzymes ciseptata, Dictyosporium elegans, Humicola cellulase, xylanase, mannanase and amylase grisea, Petriellidium boydii, Phialophora fasti- could be demonstrated for each of the giata, Phialophora hoffmannii, Phialophora species by using the different methods. The sp. A, Pseudeurotium zonatum, Rhinocla- results should be compared with the wood- diella anceps, Scytalidium lignicola, Scytali- degrading ability of the different species as dium sp. B, Wardomyces inflatus, Xylogone given in Table 2. The results of tests of the sphaerospora, Fungus A and Fungus D. growth on cellulose agar plates and the clearing of cellulose in these plates are not Ten of these species exhibited both cellulase included in Table 10. and xylanase activity in all the tests used. One of these ten species, Fungus D, was not Cellulase could be demonstrated for twenty- tested by the wood block method. It pro- four of the thirty-six species. Of these duced, however, positive results in all the twenty-four species, twenty were found to other tests. be active when the Rautela-Cowling tech- nique was employed. Twenty species pro- duced detectable amounts of cellulase in Nine of the twenty species mentioned above liquid cultures. The weight loss test with a failed to show cellulase or xylanase activity cellulose substrate (cellulose N) resulted in in one or more of the tests. Cladosporium nineteen positive results and the agar-plug sp. A did not cause any weight losses of method yielded eighteen positive results. All cellulose N and xylanase could not be species were not tested by the wood block detected in culture filtrates. This species and method but when this method was used, Scytalidium sp. B were not tested by the sixteen out of thirty species were found to wood block method. Scytalidium sp. B did be cellulolytic. Thirteen of the wood-degrad- not produce detectable amounts of xylanase ing species failed to show cellulolytic acti- in liquid cultures. Dictysporium elegans, vity on pure cellulose substrates. All of them Petriellidium boydii and Wardomyces infla- were able to produce soft rot cavities within tus did not show cellulase and xylanase the birch wood. They will subsequently be activity with the wood block method. No referred to as "non-cellulolytic7' soft rot xylanase activity was found when employing fungi. the same method for Pseudeurotium zona- turn. Cellulolytic activity could be demon- The highest number of positive results for strated only by the wood block method for xylanase activity was obtained with the Phialophora sp. A, whereas xylanase activity Rautela-Cowling technique. When this tech- was found with all the other three test nique was used, twenty-seven of the thirty- methods used. Fungus A did not produce six species were found to produce xylanase. clearing of cellulose with the Rautela-Cow- Eighteen species produced detectable ling technique nor did it produce cellulase amounts of xylanase in liquid cultures and when grown on cellulose agar plates. But this when the wood block method was used, species caused high weight losses of cellulose xylanase activity could be demonstrated for N in liquid cultures with EP medium and fourteen of the thirty species tested by this cellulase was detected in the culture filtrates. Two of the cell wall degrading enzymes could Only one cell wall degrading enzyme, viz. be demonstrated for the following species: xylanase, could be demonstrated for Cerato- Bispora betulina (cellulase and xylanase), cystis stenoceras, Cladosporium resinae, Humicola alopallonella (cellulase and xyla- Graphium sp. A and Mollisia sp. A. nase), Phialocephala dimorphospora (cellu- lase and mannanase), Phialocephala sp. A If the results given in Table 10 are compared with the wood-degrading ability of the diffe- (cellulase and xylanase) and Fungus B (xyla- rent species shown in Table 2, it is obvious nase and mannanase). Bispora betulina and that most of the wood-degrading fungi pro- Phialocephala sp. A did not show cellulolytic duced at least one of the three cell wall activity in any other test than the wood degrading enzymes studied. All the sixteen block test. Bispora betulina failed to show species which produced an erosion-type xylanase activity in this test. Both the attack (Type 2 attack) in birch wood could species produced clearing of xylan with the be shown to produce all three enzymes. RautelaCowling technique but none of them was able to produce detectable amounts of With the test methods used, five of the xylanase in liquid cultures. Humicola alopal- wood-degrading species failed to show cellu- lonella failed to show cellulase and xylanase lase, xylanase and mannanase activity. These activity in the wood block test, nor was any fungi were: Catenulari heimii, Ceratocystis xylanase produced in liquid cultures. albida, Gonatobotrys sp. A, Phialocephala sp. C and Rhinocladiella sp. A. Table 2 Phialocephala dimorphospora produced shows that these species are able to produce clearing of cellulose with the Rautela-Cow- soft rot cavities (Type 1 attack) but no ling technique but did not produce cellulase erosion-type attack (Type 2 attack) in birch when growing on cellulose agar plates. Nor wood. did this species produce any weight losses of cellulose N in liquid cultures and cellulase Of the three species Ceratocystis olivacea, could not be detected in the culture filtrates. Cladosporium resinae and Phialophora verru- This species was not tested with the wood cosa, all of which were incapable of degrad- block method. Fungus B exhibited xylanase ing birch wood, only C. resinae showed activity only in test with the Rautela-Cowling xylanase activity. Cellulase and mannanase technique. could not be demonstrated for any of them. 4 Discussion

4.1 Wood degradation and enzyme produc- of two of the components is, however, tion possible as shown by the brown rot and soft rot fungi. These fungi which degrade the Wood is degraded by a great number of cellulose and hemicelluloses of the wood different species of fungi belonging to the must produce extra-cellular and Basidiomycetes, Ascomycetes and Fungi im- hemicellulases. The main constituents of perfecti. The Basidiomycetes cause white or hemicelluloses are xylan in hardwoods and brown rot while the wood-attacking Asco- mannan in softwoods. The brown rot and mycetes and cause soft rot. soft rot fungi would consequently be expect- The term "soft rot", coined by Savory ed to produce cellulase, xylanase and man- (1954) for the attack by microfungi and nanase. A simultaneous production of xyla- characterised by cavity formation in the nase and mannanase is most probable since secondary walls of the wood cells, has in production of only one of these enzymes later years been used for all types of wood would restrict the degradation ability to degradation caused by microfungi, irrespec- either hardwoods or softwoods. tive of whether cavities are formed or not. It is interesting to note that many fungi The main constituents of wood are cellulose, which have proved to be cellulolytic have hemicelluloses and . The white rot also been found to produce xylanase and fungi decompose all three components, whe- mannanase. This also applies to fungi whose reas the brown rot fungi leave most of the wood-degrading abilities are unknown (cf. lignin as a residue. Few studies have been Lyr & Novak 1961). made on the changes in chemical composi- tion of wood attacked by pure cultures of Lyr (1963) showed that the wood-rotting microfungi. Only one species, Chaetomium Basidiomycetes, Schizophyllum commune, globosum, has been studied in detail. These Trametes versicolor, Phellinus igniarius, Col- studies were carried out by Savory and lybia velutipes, Fomes marginatus, Conio- Pinion (1958), Levi and Preston (1965) and phora cerebella, Piptoporus betulinus and Seifert (1966). Their results and scattered Gloeophyllum saepiarium produced both data from other investigations (Merrill et al. xylanase and mannanase. He also found that 1965, Bergman & Nilsson 1967 and Lund- both of these enzymes were produced by the strom 1973) indicate that the main targets cellulolytic mould Trichoderma viride and for the wood-degrading microfungi are cellu- the well-known soft rot fungus Chaetomium lose and hemicelluloses, although small re- globosum. Xylanase production by C. globo- ductions in the lignin content have been sum has also been found by Ssrensen (1952 reported. and 1957) and Fuller (1970).

A prerequisite for fungi capable of degrading Ahlgren and Eriksson (1967) showed that wood is the production of extra-cellular three wood-rotting fungi, Chrysosporium lig- enzymes that catalyze the dissolution of the noruml), Fomes annosus and Stereum san- polymeric wood components. So far as the guinolentum produced cellulase, xylanase author is aware, no fungus has proved capable of removing only one of the three l)~hefungus called Chrysosporium lignorum is, main components of wood. The removal of according to "Centraalbureau voor Schimmelcul- only one component is probably physically tures" in Baarn, identical with Sporotrichum pul- blocked by the other components. Removal verulentum Novobranova. and mannanase. Lyr and Novak (1961), who demonstrated. Five species, all producing studied the production of these enzymes soft rot cavities in birch wood, seemed to among ten species of Fungi imperfecti, lack cellulase as well as xylanase and manna- found that the three enzymes were produced nase. The apparent lack of one, two or all by all of the species. Gascoigne and Gascoig- three of the enzymes by the wood-degrading ne (1960) found xylanase activity among species is most likely due to imperfect test several species of fungi which are also known methods and not to a real incapability to to be cellulolytic. In a study of a soil fungus produce these enzymes. This will be discuss- population, Domsch and Gams (1969) found ed later in more detail. that Cephalosporium (= Acremonium) fur- catum, Chrysosporium pannorum, Coniothy- Of the three species which were unable to rium fuckelii var. sporulosum, Cylindrocar- degrade wood, viz. Ceratocystis olivacea, pon didymum, Cylindrocarpon magnusia- Cladosporium resinae and Phialophora verru- num, Doratomyces microsporus, Epicoccum cosa, only C. resinae produced xylanase. nigrum, Gliomastix (= Acremonium) muro- Cellulase and mannanase were not produced rum, Margarinomyces (= Phialophora) luteo- by any of the species. viridis, Oidiodendron echinulatum, species in the Phialophora fastigiata group, Phoma It was observed in a previous study (Nilsson eupyrena, Pseudogymnoasus roseus, Pseudeu- 1973) that all species which were able to rotium zonatum. Trichocladium opacum produce an erosion-type attack (Type 2 and Verticillium nigrescens could decompose attack) on the cell walls of birch wood were xylan. Nilsson (1973) found that all of the also able to produce clearing of Walseth mentioned species were cellulolytic and all cellulose. The same was found in the present of them, except Oidiodendron echinulatum, investigation and, furthermore, it was ascer- were able to degrade birch wood. tained that such species could also be shown to produce the enzymes xylanase and man- Thus, it is obvious that there is a connection nanase. It is obvious that hyphae which are between the ability to produce cellulase and growing in the cell lumina and which act the ability to produce xylanase and manna- upon the surface of the cell wall must nase. This is also shown by the fact that produce more or less diffusible enzymes xylanase and mannanase are often produced which accomplish the degradation of the even if the fungus is cultivated on pure components of the cell wall by means of cellulose. Eriksson and Goodell (in press), erosion. who prepared a number of cellulase-less mutants of the wood-rotting fungus Polypo- In the investigation mentioned above, it was rus adustus, found that most of the mutants also observed that a few of the species which lacked xylanase and mannanase as well. One were able to produce clearing of Walseth of the cellulase-less mutants was mutageni- cellulose did not produce- erosion-type at- zed and cellulose-degrading revertants were tack in birch wood. It was suggested that selected. These revertants were not only this failure could be due to a lack of found to be capable of degrading cellulose, wall-degrading enzymes like xylanase and but they also degraded xylan and mannan. mannanase. Another explanation was that On the basis of their results, Eriksson and the S3 layer of the wood fibres represented a Goodell suggested that the induction of resistant barrier which could not be de- cellulase, xylanase and mannanase is control- graded by these species and through which led by a single regulator gene. their enzymes could not diffuse. Acremo- nium atro-griseum and Petriellidium boydii Table 10, which summarizes the results of all are such species which failed to produce experiments in the present investigation, erosion of the cell walls of birch wood shows that all three enzymes could be although they were able to produce clearing demonstrated for twenty of the thirty-three of Walseth cellulose. The birch wood was, wood-degrading species. Five species were however, degraded by these species through shown to produce two of the enzymes and cavity formation. As may be seen in Tables 2 for three of the species only one enzyme was and 10, these species are able to produce diffusible xylanase and mannanase as well as strength of cotton duck. Domsch and Gams diffusible cellulase. Thus, it is evident that (1969) found that this species degraded their failure to produce an erosion type of xylan and CMC. Fuller (1970) found that attack is not due to a lack of these enzymes production of cellulase in liquid cultures but but is more probably due to their inability no xylanase was found. King and Eggins to degrade the S3 layer. (1973) showed that the fungus reduced the strength of filter paper. A literature survey covering the degradation of cell wall components by those species in Phialophora verrucosa. Reese et al. (1950) the present study which are fully identified found that this species was unable to reduce has been made. For several of the species no the strength of cotton textile. information about cellulolytic activity or degradation of xylan and mannan could be Pseudeurotium zonatum. Domsch and Gams found. Some of the data collected is found (1969) showed that this species degraded below. The data presented by the author in a xylan and CMC. previous paper (Nilsson 1973) are not in- cluded. Scytalidium lignicola. King and Eggins (1973) showed that this fungus could de- Ceratocystis albida (= Ophiostoma albidum). grade cellulose. Kaarik (1960) found that cellulose was a very poor source of carbon for this species, as well as for other Ceratocystis species. If the above data are compared with the However, she stated that all species were results obtained in the present study, it is "able to make a starvation type of growth evident that there is good agreement. Un- on it". fortunately, no studies seem to have been done earlier with the most interesting species Chrysosporium pannorum. Degradation of in this study, i.e. those which degrade wood filter paper by this species was found by but fail to show production of the necessary Schaefer (1957). Domsch and Gams (1969) enzymes. The only exception is the study of found that the fungus degraded xylan and Ceratocystis albida by Kaarik (1960). CMC. Production of amylase is not necessary for Cladosporium resinae. Some variation seems the degradation of wood but might be to exist among different strains. Parbery beneficial for fungi colonizing wood sub- (1969) found that one out of five isolates strates since starch is an important reserve was able to degrade cellulose. nutrient in many trees. The present investi- gation shows that the ability to degrade Coniothyrium fuckelii var. sporulosum. starch is common among wood-inhabiting Domsch and Gams (1969) found that this microfungi. This was also found by King and fungus degraded xylan and CMC. Eggins (1973) who found amylolytic activity in all of the thirty-three different mould and Humicola alopallonella. Meyers and Rey- staining fungi which were tested. nolds (1959) found that this species was cellulolytic. 4.2 Test methods and results Humicola grisea. Traaen (19 14) already showed that this fungus decomposed cellu- In the following the results obtained with lose. Later, several investigations have prov- the different test methods will be discussed. ed the cellulolytic activity of this species (cf. Domsch & Gams 1970). Poole and Taylor Numerous methods have been used for (1973) found production of cellulase and assays of cellulolytic activity among micro- xylanase. organisms. Eriksson (1969) gives a list of various assay methods used for determining Phialophora fastigiata. Reese et al. (1950) cellulolytic activity. His list contains the showed that P. fastigiata reduced tensile following methods: loss in weight of insoluble substrates truly cellulolytic fungi possess an additional decrease in mechanical properties of enzyme, C1, which enables them to degrade fibres or films native cellulose. King and Vessal (1969) also change in turbidity of cellulose suspen- defined the C1 enzyme as an enzyme which sion is required for the hydrolysis of highly increase in reducing ends groups oriented solid cellulose, like cotton and decrease of viscosity of cellulose deriva- Avicel, by 0 - 1 +4 glucanases (=C,). Since tives wood cellulose also is a highly oriented solid colorimetric determination of dissolved cellulose, it follows from the definition that decomposed products of cellulose all fungi which are able to degrade wood measurements of clearance zones in cellu- must produce C1 enzymes. Thus, the inabil- lose agar ity of certain fungi to degrade cotton or Avicel does not necessarily mean that they To this list may be added: lack C1 enzymes. It may possibly be that the 8) microscopic observations of morphologi- culture conditions are unsuitable for the cal changes in a cellulose substrate (like production of this enzyme. fibres or cellophane) Whether the degradation of a modified 9) growth on cellulose agar cellulose such as Walseth cellulose indicates C1 activity or merely C, activity has already Except for method 9, these methods can be been discussed in a previous paper (Nilsson used both for determining the cellulolytic 1973). The findings, that most of the fungi activity of growing microorganisms and the which degraded Walseth cellulose were also activity of isolated cellulases. able to degrade wood cellulose, and the fact that CMC-degrading fungi failed to degrade Methods 3 and 7 are very much of the same Walseth cellulose, support the former type. These assay methods, which aim at a clearing of cellulose substrates, have been theory. extensively used in this study. Methods 1, 8 and 9 have also been used. All of the As can be seen in Table 10, a high number of positive results were obtained by means of methods referred to may be considered to be indicative of a C1 enzyme sensu, Reese, Siu the Rautela-Cowling technique. This techni- and Levinson (1950). que has several advantages. It is simple to carry out and, as pointed out by Rautela and Cowling (1966), cellulolytic activity is Measurements of the decrease of viscosity of "determined directly on a continuous, cellulose derivatives (method 5) have been cumulative basis". Other substrates than widely used for determining the cellulolytic cellulose, e.g. hemicelluloses and starch, can activity of microorganisms. Most of the also be tested with this technique, as has studies have been carried out with carboxy- been done in the present study. This method methyl cellulose (CMC) as substrate. Yet also seems to be quite sensitive and appears Reese and Levinson (1952) already found to give positive results even for species with several non-cellulolytic organisms that pro- weak cellulolytic activity. duced CMC-degrading enzymes. These enzy- mes are usually referred to as C, enzymes. It was already shown in a previous paper, The production of C, enzymes by non-cellu- Nilsson (1973), that cultivation on medium lolytic fungi has later been demonstrated VII suggested by Bravery (1968) yielded a in other investigations. Wood (1969) re- higher number of positive results for cellu- ferred to these fungi as "pseudo-cellulolytic" lolytic activity than cultivation on the me- species. The aforementioned method has dium formulated by Rautela and Cowling applications in the studies of the compo- (1966). Several species which failed to pro- nents of the cellulase system, but it should duce clearing on R-C medium did clear the not be used for assays of the cellulolytic cellulose on B-VII medium. This was also activity of microorganisms. confirmed in the present study in the case of two additional species, viz. Cladosporium sp. Reese, Siu and Levinson (1950) suggested that A and Fungus D. This phenomenon might be explained by the presence of an alternative porosity. Both are factors which will in- carbon source (yeast extract) in the R-C crease the susceptibility to hydrolysis (cf. medium, since it was demonstrated by Norkrans 1950, Walseth 1952 and Stone et Bravery (1968) that small amounts of alter- al. 1969). native carbon sources, like asparagine and yeast extract, inhibited clearing of cellulose Table 3 shows the results of three different agar by certain soft rot fungi. The decreased assay methods of cellulolytic activity; amount of clearing of cellulose produced by 1) growth on cellulose agar, 2) clearing of all active species on medium R-C compared cellulose underneath or around fungal colo- with the clearing that occurred on B-VII nies growing on cellulose agar plates and medium (see Table 2) might also be due to 3) clearing of cellulose in test plates around inhibition by the presence of yeast extract in transferred mycelium-agar plugs. The only the former medium. Alternative carbon difference between method 2 and the sources like glucose, asparagine and yeast Rautela-Cowling technique is that the extract appear, however, to exert a varying former is carried out in petri dishes while the influence on the cellulolytic activity de- latter employs test tubes. pending on the species of fungi and also on It is evident from the results that growth on the culture conditions. As can be seen from cellulose agar is a very uncertain criterion for the results of the tests with cellulose in cellulolytic activity. Visual estimation of the liquid cultures (Tables 6 and 7), the presence amount of growth of fungi on agar is of asparagine and yeast extract in the EP difficult since the fungi might produce dif- medium appeared to have no adverse effects ferent types of colonies. Some fungi produce on the cellulolytic activity of the tested a dense mycelium on a restricted area on a fungi. Park (1973), who made a study certain medium, while on other media the similar to that of Bravery (1968), tested same species will produce a widely spreading several modifications of the Eggins and Pugh but thin mycelium. The estimation of growth (1962) medium. He reached the conclusion and non-growth on cellulose agar media of that the presence of asparagine might have the types used in the present investigation disadvantages, whereas yeast extract seems seems impossible since the truly non-cellu- to have no disadvantages. Dennis (1972), lolytic fungi also produced some growth on who studied the clearing of cellulose agar by the B-VII medium which contains no yeasts ( spp.) found that small source of carbon other than cellulose. It is amounts of yeast extract and asparagine possible that these fungi are able to make stimulated the activity of some of the some growth on impurities containing car- isolates, but reduced the activity in others. bon, which may be present in the Avicel that Norkrans and Aschan (1953), who cultivated was used as cellulose substrate. The other different strains of Collybia velutipes on a cellulose agar medium used, F6A, contains weak yeast-glucose-cellulose medium, found both glucose and yeast extract. This explains that the cellulolytic activity did not change why all of the tested species were able to much when cultivated on a richer yeast grow on this medium. In fact, two of the extract medium. If the strains were culti- non-cellulolytic species grew better on F6A vated on a glucose-free medium, the activity celluloses agar than some of the cellulolytic was the same or decreased. species. The species which had failed to produce clearing of cellulose with the It is obvious from the results presented in Rautela-Cowling technique (see Table 2) Table 2 that the amount of clearing of made sparse or moderate growth even on the cellulose with the Rautela-Cowling techni- F6A cellulose agar. They did not appear to que depends very much on the type of be able to utilize the cellulose in the cellulose substrate. The greatest amount of medium, since no clearing of the cellulose clearing was obtained on Walseth cellulose could be detected in the agar nor was any and ball-milled cellulose. The preparation of cellulase detected with the mycelium-agar these two types of celluloses involves treat- plug technique of Savory et al. (1 967). ments that increase the proportion of amorphus cellulose and also the intrinsic A rather paradoxical conclusion which can be drawn from the results obtained in the investigation. With the other test methods present study is that cellulose agar substrates nothing is known about the formation of are not suitable for the isolation of certain cellulase on the substrate used. But when cellulolytic fungi. solid wood is used as substrate, the degrada- tion of the wood cell walls in the form of The two agar media used for these studies erosion or cavity formation indicates that contained Avicel, a cellulose which is more cellulase is present. The degradation of the resistant to hydrolysis than Walseth cellulose cellulose in the cell walls is easily detected and ball-milled cellulose. This might explain by microscopic studies using polarized light the failure of most of the species to produce and is in itself a demonstration of cellulo- visible clearing in the cellulose agar on which lytic activity. By means of the wood block they grew. It was already shown in Table 2 method it could be shown that the cellulase that clearing of Avicel was very weak, as present in the wood could degrade a pure compared with the clearing of the two other cellulose such as Walseth cellulose (see Table celluloses. But the obscuring by dense 4). mycelia, which made it difficult to detect any clearing probably also contributed to The wood block method was the only means the low number of positive results. by which cellulase could be demonstrated for Bispora betulina, Phialocephala sp. A and The agar-diffusion method employing myce- Phialophora sp. A. The xylanase activity of lium-agar plugs adopted after Savory et al. Ceratocystis stenoceras was demonstrated (1967) had no advantages over the Rautela- only by this method. Fungus A, which had Cowling technique. The agar-diffusion failed to demonstrate cellulolytic activity in method is more time consuming and the the previous tests discussed, was found to be assays of cellulolytic activity are made after cellulolytic when the wood block method certain arbitrarily chosen times. The number was used and also when cultured on cellulose of positive results was lower with this in liquid media. method than with the Rautela-Cowling technique (see Table 10). Cellulolytic activity could not be detected with the mycelium- The species Catenularia heimii, Ceratocystis agar plug technique for two of the fungi, albida, Ceratocystis stenoceras, Gonato- Phialocephala dirnorphospora and Rhinocla- botrys sp. A, Graphiurn sp. A, Mollisia sp. A, diella anceps, which had produced clearing Rhinocladiella sp. A and Fungus B, all of which failed to show cellulolytic activity of cellulose with the Rautela-Cowling tech- with the other test methods used, also failed nique. with the wood block method. However, If the production of cellulase on the cellu- microscopic studies showed that numerous lose agars F6A and B-VII is compared for cavities were present in all the wood blocks the fungi which produced clearing of cellu- attacked by these species,. with the excep- lose only on medium B-VII when the tion of wood blocks attacked by Mollisia sp. Rautela-Cowling technique was used (see A in which only a few cavities were found. It Table 2), it is evident that the glucose, and was further ascertained that several species possibly also the yeast extract, present in the which showed celluiolytic activity in other cellulose agar F6A did inhibit the cellulo- tests failed in the wood block method. Two lytic activity of these fungi. Of these fungi explanations can be given for the failures in only Cladosporiurn sp. A was able to pro- the wood block method: 1) the enzymes duce detectable amounts of cellulase on F6A are produced in only minute amounts in the agar. wood, adequate for wood degradation but not sufficient for diffusion out into the test The test method described in Materials and agar; 2) the enzymes are firmly bound methods (section 2.5) which employs the either to the wood substrate and/or the detection of cellulase and xylanase present mycelium and are thus not able to diffuse in wood blocks attacked by the test organ- freely out into the test agar. isms, is based on different premises than the other test methods used in the present The method in which the fungi were cul- tured on cellulosic substrates in liquid media susceptibility of a certain cellulose substrate provided two criteria for cellulolytic activ- not only depends on the structure of the ity. The first was weight loss of the substrate substrate but also on the medium in which and the other detection of cellulase in the the tests are carried out. The order of culture filtrate. A weight loss of the cellu- increasing susceptibility of different types of losic substrate is not necessarily correlated cellulose under one condition might there- with detectable amounts of cellulase in the fore change drastically under other condi- culture filtrate. This can be seen in Table 7 tions. where, for example, Acremonium atro- griseum, Cordana pauciseptata and Scytali- Differences in the distribution of the cellu- dium lignicola is shown to have caused high lose substrates within the nutrient solution weight losses although no cellulase could be are also likely to influence the results. detected in the culture filtrates. This might Cotton, for example, is partly floating and be explained by binding of the enzyme to partly immersed in the solution, while the the mycelium and/or to the substrate. The Avicel particles are completely immersed. same fungi, however, produced detectable These particles will sink to the bottom of cellulase on medium EP. Medium B-VII-L the flasks if stationary cultures are used. is a rather poor medium compared with Thus, apparent differences in the susceptibil- medium EP. Medium EP contains yeast ity of the cellulose substrates as found here extract and 2 12 mg N/liter while medium when growing fungi in liquid cultures must B-VII-L lacks yeast extract and contains not be considered to reflect an absolute only 115 mg N/liter. When growing on indication of the susceptibility of these medium B-VII-L the fungi must use their substrates to fungal degradation. enzymes more economically than when growing on the richer medium. Free en- It was expected that the species which zymes in the solution can be regarded as a produced clearing on B-VII but failed to wastage of available resources, especially produce clearing of cellulose on the R-C nitrogen. medium (see Table 2) would give the best results on B-VII-L medium since this The fact that cellulolytic enzymes are de- medium lacks alternative carbon sources. tected in the culture filtrates is not always But of these species both Cladosporiurn sp. correlated with significant weight losses of A and Humicola alopallonella produced the substrate. Table 6 shows that cellulase better results on the EP medium. Only was detected in culture filtrates from Cla- Scytalidium sp. B showed a positive response dosporium sp. A on cellulose N, Dictyospo- to medium B-VII-L. The same positive rium elegans on Avicel and cotton and response was found for Rhinocladiella Phialophora fastigiata on Avicel while no anceps, although this species was also able to significant weight losses occurred. Thus the produce clearing of cellulose on the R-C use of two methods for determining cellu- medium. lolytic activity increased the number of positive results. If the results from the experiments with liquid cultures are compared with the results The differences in nitrogen content between obtained with the Rautela-Cowling tech- the two media might explain why the weight nique, it is found that essentially the same losses were generally lower on the B-VII-L fungi have shown positive results by the two medium. However, two species, Acrerno- test methods. There are only two excep- nium atro-griseurn and Cordana paucisep- tions: one is Phialocephala dimorphospora tata, caused higher weight losses of one of the which failed to give any indications of substrates, viz. cotton, on medium B-VII-L cellulolytic activity when cultured in liquid than on medium EP. It is difficult to explain media; the other was Fungus A which had why the weight losses of the other two failed to produce clearing of cellulose with substrates, Avicel and cellulose N, were the Rautela-Cowling technique. This fungus lower on the B-VII-L medium than on produced significant weight losses in liquid medium EP. The results show that the cultures and cellulase was found in the culture filtrates. No explanation has been liquid cultures and both cellulase and xyla- discovered for the behaviour of Fungus A, nase were found in the culture filtrates. The but the physiological conditions are probab- purpose of using birch wood meal as a ly not suitable for the production of cellu- substrate was that it contains all the consti- lase when the fungus grows on agar. tuents of solid birch wood, in which several of the test organisms made their only mani- With the exception of Fungus A, all soft rot festation of cellulolytic activity in the form fungi which failed to produce clearing of of cavities. But even the cavity-forming cellulose with the Rautela-Cowling tech- species tested were unable to degrade the nique also failed when cultured in liquid birch wood meal and to produce cellulase and media on cellulose substrates. The con- xylanase. clusion which can be drawn is that these species did not produce cellulase under the It is a common observation that lignified prevailing conditions. It might be argued substrates are less susceptible to degradation that cellulases were not detected in the than non-lignified substrates. This seems culture filtrates because these species may especially to be the case when isolated have only cell-bound enzymes. But even if cellulase systems are used. However, Table 8 the enzymes are cell-bound, attack on the clearly shows that some species of micro- cellulose would still be possible and this fungi are able to degrade a lignified fibre would lead to a weight loss of the substrate. more readily than a non-lignified fibre. However, no weight losses were recorded for Ceratocystis albida is an exemple of such a the species mentioned. species. This fungus failed to degrade the cotton fibres while the jute fibres were The high numbers of positive results for degraded to a significant degree although cellulolytic activity obtained from the tests jute fibres contain about 11 percent lignin on cellulose in liquid cultures, shown in (Roelofsen 1959). Wardomyces inflatus was Table 10, were achieved only by the use of able to degrade cotton but the weight losses two nutrient media of different composi- of jute were higher. Only Xylogone sphae- tion. If only medium EP had been used the rospora caused higher weight losses of cot- number of positive results from the weight ton than of jute. Of the five species used in loss determinations would have been 17 this experiment, all except X. sphaerospora instead of 19 and cellulase would have been were able to produce soft rot cavities in detected in the culture filtrates for 18 birch wood. Under the conditions employ- instead of 20 species. The number of posi- ed here, Ceratocystis albida and Wardomyces tive results would have been far less if only inflatus were also able to produce cavities in medium B-VII-L had been used. When the the lignified jute fibres but not in the Rautela-Cowling technique was used, all non-lignified cotton fibres. positive results could be obtained on a single medium, viz. medium B-VII. It is obvious C. albida, which evidently is unable to that this technique is preferable to tests on produce cellulases in liquid cultures on pure cellulose in liquid media if the objective is a cellulose substrates (cf. Tables 6 and 7), survey of the cellulolytic activity of micro- could thus not degrade the cotton fibres. fungi. Wardomyces inflatus which is able to pro- duce cellulases under the same conditions (cf. Birch wood meal in liquid cultures appears Tables 6 and 7) degraded the cotton fibres in to be an unsuitable substrate for tests of this experiment but the degradation of the cellulolytic activity since no activity was jute fibres was more extensive due to pro- obtained, even with the highly cellulolytic minent cavity formation in these fibres. species Chrysosporium pannorum. Degrada- tion of the substrate did not occur, neither Bispora betulina and Phialophora sp. A are could cellulase nor xylanase be detected in able, as will be shown in a later paper, to the culture filtrates. As can be seen in Table produce cavities in jute fibres. However, 6, the same fungus caused an extensive cavities are not formed by these species in degradation of Avicel and cellulose N in fibres which are immersed in a nutrient

29 solution as they were in the present inves- Ceratocystis albida and Phialocephala sp. A tigation. Both of the species are also unable (see Table 9). Thus, the initial lack of an to produce cavities in cotton fibres in liquid easily available carbon source does not ex- cultures. Cellulase was not produced when plain why these species failed to show they were cultured in liquid cultures on pure cellulolytic activity. cellulose substrates or on jute fibres (cf. Tables 6, 7 and 8). These facts explain why The addition of 0.1 percent glucose to the two fungi failed to degrade the cotton medium EP delayed cellulase production of and the jute fibres. Fungus A only slightly. Since the EP medi- um also contains small amounts of aspara- Both the cotton and jute fibres were degrad- gine and yeast extract it is evident that this ed by Xylogone sphaerospora by a type of species is not prevented from producing erosion attack. No soft rot cavities were cellulase by small amounts of alternative formed in any of the fibres. The erosion is carbon sources. caused by enzymes which are released into the liquid medium by the fungus. The The cellulase production of Cladorrhinum breakdown of the jute fibres under these sp. A and Xylogone sphaerospora was delay- circumstances appears to be limited by the ed by glucose additions and Cladorrhinum presence of lignin. The fungus consequently sp. A produced no cellulase at a glucose caused the highest weight losses on the concentration of 0.5 percent. It has been non-lignified cotton fibres. shown by Bemiller et al. (1969) and others that the cellulase production does not start The observations made in the experiments until the glucose in a nutrient solution has discussed above might also explain the been consumed. The more sugar added, the results obtained by Chakravarty et al. longer time it will take for the consumption (1962). They compared the degradation of of glucose and the initiation of cellulase jute fibres with degradation of filter paper. production. This fact will also explain the All species tested except one, Sordaria hypo- delay in cellulase production observed in the coproides, caused the highest weight losses experiments carried out here. when cultured on filter paper. S. hypoco- proides, however, caused higher weight loss of The conclusion drawn from the experiments jute fibres than of filter paper. It might be presented here and from the preceeding possible that this species is also able' to discussion is that there is no single simple produce cavities within the jute fibres and method for the assay of the cellulolytic that the amount of cellulase released into activity fo microfungi. This does not apply the nutrient medium is small. But so far as solely to fungi isolated from wood, since the present author is aware, no studies of the fungi with an apparent lack of cellulolytic cavity-forming ability of this fungus have activity but which are able to produce been carried out. cavities in various cellulosic substrates, might also be isolated from other sources, for Simpson and Marsh (1964) discovered that example soil and compost heaps. It has been Aspergillus niger and certain other Asper- shown by Baker (1939) and by Nilsson gillus species caused a strength loss of cotton (1974) that in nature cavity formation of fabric only when a small amount of glucose the soft rot type is not restricted to wood was added to the test medium. Similarly, but might also occur in other materials Basu and Ghose (1 960) found that additions containing cellulose fibres. of sugar, especially xylose, stimulated the production of cellulase by certain species of If a total survey of the cellulolytic activity is fungi which had been found to be unable to objective, the following procedure is recom- produce cellulase when growing on pure mended: cellulose. The addition of small amounts of glucose as "start-glucose" to medium EP 1) All the isolated species should be tested had, however, no positive effect on the by means of the Rautela-Cowling tech- production of cellulase by Bispora betulina, nique, employing Walseth cellulose as the substrate. 0.25 percent appears to be a The test method described above may suitable concentration of the cellulose. appear tedious and time-consuming but until Ball-milled cellulose might also be used new methods for assaying cellulolytic activi- but the number of positive results is ty are found, it is a procedure which will likely to decrease. Avicel and similar give reliable information of the cellulolytic types of celluloses should be avoided. The activity of species of microfungi. use of medium B-VII is recommended since it yielded a greater number of positive results than the medium used by 4.3 The anomalous behaviour of the "non- Rautela and Cowling (1966). All of the cellulolytic" soft rot fungi species which produce clearing of the cellulose can safely be regarded as cellulo- It was previously suggested that the wood- lytic but obviously nothing is known degrading species would produce all the about the cellulolytic activity of the three cell wall degrading enzymes cellulase, remaining species since certain soft rot xylanase and mannanase. This assumption fungi of the type described in the present was also confirmed for about half of the study will not produce any clearing. number of the species tested. But some wood-degrading species failed to exhibit one 2) All of the species which have failed to or more of these enzymes when assayed with produce clearing of cellulose are tested on the test methods employed. Various expla- birch wood (Betula species) for the abili- nations of this behaviour are possible. The ty to form soft rot cavities. Some other lack of activity against xylan and mannan hardwood species might equally well be could be due to alterations of these substra- used, such as aspen (Populus species) or tes during their preparation from wood so beech (Fagus silvatica), but it must be that their structure differs too greatly from ascertained that the wood used is not the natural. It is also possible, in fact, that very resistant to attack by soft rot fungi. some fungi lack these enzymes. It is more The decay method used by Nilsson difficult to explain the failure of certain (1973) and which employs agar slopes in species to degrade the pure cellulose sub- test tubes is recommended. The time strates. This phenomenon and others con- required for cavity formation varies cerning the production of cellulase will now among the species. Most of the species be discussed in more detail. tested here have formed numerous cavi- ties in birch wood already within 2-4 Cellulase is the only enzyme which is with weeks. The species which produce soft certainty known to be produced by the rot cavites must also be regarded as wood-degrading species since degradation of cellulolytic and be included in the total cellulose occurred in the birch wood fibres. number of cellulolytic species. It is highly This degradation was easily detected in the probable that most of the remaining microscope using polarized light. Since no species are non-cellulolytic, though it chemical analysis of birch decayed by the cannot be totally excluded that some of test organisms was made, nothing is really these species are able to degrade cellu- known about the degradation of xylan. It lose under certain special conditions. may, however, be assumed to be very prob- The procedure described above will, able that the xylan was also decomposed. nevertheless, increase the number of cel- lulolytic species, even if not all of the The discussion will be focused on the follow- cellulolytic species are detected. ing organisms, all of which produced soft rot cavities in the birch wood but failed to The recommended procedures are restricted exhibit cellulolytic activity on pure cellulose to assays of the cellulolytic activity of substrates: Bispora betulina, Catenularia hei- microfungi. Problems concerning the assays mii, Ceratocystis albida, Ceratocystis steno- of the cellulolytic activity of the basidio- ceras, Gonatobotrys sp. A, Graphium sp. A, mycete fungi will be discussed in a later Mollisia sp. A, Phialocephala sp. A, Phialoce- paper. phala sp. C, Phialophora sp. A, Rhinocladiel- la sp. A and Fungus B. These fungi have blocks in laboratory tests are most heavily previously been referred to as "non-cellulo- degraded in the outer layers. Weight losses lytic" soft rot fungi. The presence of cellula- reported from laboratory tests refer to the se in attacked birch wood blocks could be weight loss in the whole blocks. Thus, a thin demonstrated for some of these species. The outer layer of the wood blocks might be experiments carried out using the Rautela- very highly degraded even if the total loss in Cowling technique and the cultivation of the weight is small. Microscopic observations of fungi on pure cellulose substrates in liquid the soft rot attack on birch wood blocks by media showed that no cellulase was produc- the "non-cellulolytic" soft rot fungus Cera- ed under the prevailing conditions. These tocystis albida (Nilsson, not published) have findings suggest that cellulase of the men- shown that the wood fibres in the outer tioned species is an inducible enzyme and layers of the birch wood blocks may already that the induction is regulated by factors be degraded to 50 - 90 percent after three which are so far unknown. weeks. Thus the low weight losses caused by some soft rot fungi seem to a higher degree Various explanations may be advanced for to be due to a restriction of the attack to the the failure of the "non-cellulolytic" soft rot outer layers of the wood than to a low fungi to degrade pure cellulose substrates cellulolytic activity. under the conditions prevailing in the pre- sent investigation. 2) The cellulase production is temperature- dependent. 1) the "non-cellulolytic" soft rot fungi have a very weak cellulolytic activity. It has been demonstrated that the cellulolytic activity of some fungi is quite sensitive to There have been some false opinions regard- temperature. Marsh et al. (1949) and Reese ing the cellulase activity of the soft rot fungi and Levinson (1952) have shown that Cla- (cf. Lyr 1960, Levi 1964 and Wilhelmsen dosporium herbarum has cellulolytic activity 1965). It has been claimed that these fungi at 25'~ but not at 30'~. Hirsch (1954) produce less of these enzymes than the found that Neurospora crassao had a very white and brown rot fungi. Such generalisa- weak cellulolytic activity at 25 C compared tions should not be drawn unless they are with the activity at 35'~.The wood-degrad- based on a large number of observations. ing species in the present investigation, Seifert (1966) could, in fact, show that in which had failed to produce clearing of the early stages, cellulose degradation of cellulose at the ambient room temperature beech wood by the soft rot fungus Chaeto- (23-25') were also incubated at 15'~and mium globosum was greater than the degra- 30'~. Since no clearing of the cellulose dation caused by one white and one brown occurred even after incubation for six weeks rot fungus. at these temperatures, it must be regarded as unlikely that the failure to produce clearing In fact, the soft rot species tested here can zones in the cellulose was due to a tempera- be regarded to have a rather high cellulase ture factor. activity when growing on birch wood. If a typical soft rot cavity is observed, only a 3) Some growth factors are lacking in the single is present in each cavity. The media used. cavity may be very large in relation to the size of the hypha. This indicates a high The "non-cellulolytic" soft rot fungi produc- enzyme activity per mycelium unit. ed at least some growth on the cellulose agar media used. All species also grew in the The opinion that the soft rot fungi have a liquid media EP and B-VII-L when glucose low cellulolytic activity probably is a result was added. Thus, it is not likely that their of the fact that several of these fungi cause failure to show cellulolytic activity is due a comparatively small weight losses of the lack of growth factors, unless certain growth wood blocks used in decay tests. It has been factors are required specially for the pro- shown by Lundstrom (1973) that wood duction of cellulase. 4) The "non-cellulolytic" soft rot fungi ini- tions used, it is unlikely that cell-bound tially require an easily available carbon enzymes is an explanation of the anomalous source ("start-glucose") behaviour of these fungi. Furthermore, the clear zones obtained around wood sections This hypothesis was tested (see Table 9) and cut from wood blocks decayed by some of it was found that no stimulation of the the "non-cellulolytic" species indicate that cellulase production could be ascertained the cellulases of these species are not cell- when glucose was added in small amounts. bound.

5) The cellulase is cell-bound. 6) Cellulase is not produced on pure cellulo- According to Cowling and Brown (1969), se. the cavities formed in the wood cell walls by soft rot fungi are an example of a degrada- The cellulose substrates which were used, tion pattern produced by cell-bound enzy- viz. Avicel, cotton, cellulose N, Walseth mes. However, the mere fact that the degra- cellulose and ball-milled cellulose, can be dation is restricted to localized areas within regarded as pure celluloses containing only the cell walls does not prove that the small amounts of other substances. Wood, enzymes are cell-bound. The restriction of on the other hand, contains hemicelluloses the attack is more probably due to the and lignin in addition to cellulose. It might compact structure of the cell wall in which be possible that the hemicelluloses and/or the enzymes are formed. If the enzymes are lignin in some way stimulate the production unable to diffuse through this compact of cellulase. Basu and Ghose (1960), have, structure they will migrate only with the for example, found that several species of simultaneous dissolution of the components microfungi were able to produce cellulase on of the cell wall. jute holocellulose (which contains hemicellu- loses) but not when grown on a pure In the present study it has been found that cellulose like filter paper. several of the fungi which produce soft rot cavities also produce diffusible cellulases as In the present study some of the "non-cellu- indicated by the clear zones formed in lolytic" soft rots were grown on birch wood cellulose agar beneath the growing fungi. meal and jute fibres. As mentioned previous- Several of these species also release cellulase ly, birch wood meal was not degraded by into the nutrient solution when grown in any of the "non-cellulolytic" soft rot fungi liquid media. Most, but not all, of these and the jute fibres were degraded only by species also cause an erosion of the wood one species, viz. Ceratocystis albida. This cell walls. However, it might be possible that would seem to indicate that C. albida is able such species produce two different types of to degrade cellulose only when it is associa- cellulases; one which is diffusible and pro- ted with hemicellulose and lignin. This is, duced by luminal hyphae, while the other is however, not the case as will be shown in a cell-bound and produced by hyphae within later paper. As stated earlier, the use of the cavities in the wood cell walls. The wood meal and jute fibres in liquid media is "non-cellulolytic" species which were all an unsuitable method for assays of enzyma- found to be unable to cause an erosion of tic activity since even a cellulolytic species the wood cell walls would thus only produce like Chrysosporium pannorum failed to the cell-bound enzyme. show any activity on birch wood meal. Thus, the experiments carried out in the present As stated previously, the possession of only investigation are not suitable for studies of cell-bound cellulases would still allow the the influence of associated substances on the "non-cellulolytic" species to degrade the production of cellulase. These questions will celluloses used as substrates in the liquid be discussed more in detail in a later paper. cultures as well as the cellulose used in the agar media. Since none of these species were 7) Cellulase production is regulated by the able to degrade cellulose under the condi- physical structure of the wood. The formation of soft rot cavities in wood rot fungi: Theses species are induced to must be regarded as a rather unique type of produce cellulase when their hyphae are degradation. The enzyme-producing hyphae growing parallel to the cellulose microfibrils of white and brown rot fungi and the whithin the wood cell walls. hyphae of microfungi which cause erosion of the wood cell walls, are more or less random- 8) The physiological conditions of cultiva- ly distributed within the wood. The distri- tion have not been suitable for cellulase bution of the cavity-forming hyphae of the production. soft rot fungi are, however, closely related to the structure of the wood cell wall. These A common feature for all types of experi- hyphae always appear to grow parallel with ments carried out here is that the cellulose the direction of the cellulose microfibrils substrates have had a very high moisture within the cell walls (cf. Bailey & Vestal content. The cellulose has either been im- 1937 and Nilsson 1974). mersed in liquid media or suspended in agar substrates. Since no experiments with cellu- It was suggested in a previous paper concern- lose substrates with lower moisture content ing the formation of soft rot cavities in have been carried out in the present study, it various cellulose fibres by Humicola alopal- is impossible to discuss the influence of the lonella (Nilsson 1974) that the cellulase moisture content at the present stage. It will, production was stimulated when the hyphae however, be shown in a later paper that the grow parallel to the cellulose microfibrils moisture content of the cellulose substrates inside the wood cell wall. This idea was has a decisive influence on the degradation supported by the following observation: The of these substrates by certain soft rot fungi. fungus, when offered a thin transverse sec- tion of birch wood, failed to produce an If the last part of the discussion is summari- erosion attack on the exposed secondary cell zed,, the following conclusions can be drawn: walls. This indicated a low cellulase activity a) The inability of the "non-cellulolytic" of the hyphae which grew over the wood soft rot fungi to degrade pure cellulose is section. The only hyphae which exhibited not due to 1) weak cellulolytic activity; cellulolytic activity were those which pene- 2) temperature-dependent cellulase in trated the secondary cell walls in the longi- the sense that this enzyme is produced tudinal direction of the wood fibres. These only within a certain temperature range; hyphae appeared to follow the direction of 3) lack of growth factors; 4) lack of the cellulose microfibrils and they formed "start-glucose"; 5) cell-bound enzymes. typical soft rot cavities in the cell walls. b) The failure of the "non-cellulolytic" soft The only exhibition of cellulolytic activity rot fungi to degrade pure cellulose might of the "non-cellulolytic" soft rot fungi be due to: 1) The absence of substances which was found was the formation of soft such as hemicellulase and lignin which are rot cavities in the secondary cell walls of associated with the cellulose in wood; birch wood. The hyphae within the cell 2) specific regulation of the cellulase walls, which secrete cell wall degrading production by the physical structure of enzymes and give rise to the cavities, appear the wood; 3) unsuitable physiological to follow closely the direction of the cellulo- conditions. se microfibrils in the same manner as Humi- cola alopallonella. Thus, there is a strong The data from the experiments carried out relationship between the enzyme-producing in the present investigation are evidently (= cavity-forming) hyphae and the physical insufficient to provide a solution of the structure of the wood cell walls. Based on problem discussed. It is obvious that much these observations, the following hypotheti- work remains to be done before the decay cal explanation is suggested for the anoma- mechanisms of the soft rot fungi can be lous behaviour of the "non-cellulolytic" soft understood. Summary

The production of the enzymes cellulase, Among other factors, studies were carried xylanase, mannanase and amylase have been out to determine the effects of the composi- studied for thirty-six different species of tion of different media, additives of small wood-attacking microfungi. The ability of amounts of glucose (as "start-glucose"), the the fungi to degrade wood was already type of cellulose substrate, different cultiva- known. Three of the species lacked the tion conditions such as shake and stationary ability to degrade birch wood while the cultures and different incubation tempera- remainder gave rise to the following three tures. The results showed that the composi- patterns of attack in birch wood: 1) soft rot tion of the cultivation media was of decisive cavities in the secondary cell walls (= Type 1 importance for certain fungi. Small amounts attack); 2) erosion of the cell walls caused of yeast extract had an inhibiting effect on by fungal hyphae in the cell lumina (= Type cellulase activity during cultivation on cellu- 2 attack); and 3) simultaneous attacks of lose agar media. This effect was not notice- Type 1 and Type 2. able during cultivation on a cellulose sub- strate in liquid solutions. The largest clearing It was presumed a priori that the wood- zones were obtained on Walseth cellulose degrading microfungi produced all of the and ball-milled cellulose. Significantly smal- three cell wall degrading enzymes, cellulase, ler clearing zones were obtained on Avicel xylanase and mannanase. No effort was made and on cellulose treated with hydrochloric to obtain an absolute measurement of the acid. The different species of fungi reacted various enzyme activities. The investigations individually when cultivated in shake or were therefore concentrated on an endea- stationary cultures. Some species caused the vour to demonstrate the presence of the greatest losses in weight during cultivation in different enzymes. shake cultures while others showed greater activity during cultivation in stationary cul- The enzyme activity of the fungi was studied tures. by means of the following methods: 1) mea- suring the clearing zones under fungal cultu- All of the fungi studied, with the exception res growing on various substrates on agar of Phialophora verrucosa, could degrade columns in test tubes, Rautela-Cowling tech- starch. Among the wood-degrading fungi, all nique (cellulase, xylanase, mannanase and of the three cell wall degrading enzymes, amylase activity); 2) the growth, formation cellulase, xylanase and mannanase, could be of clearing zones and production of cellulase determined in twenty species. Four of the in fungal cultures on cellulose agar plates; twenty species only gave rise to Type 1 3) determination of the occurrence of cellu- attacks (cavities), while the remainder caus- lase and xylanase in birch wood which had ed some attack of Type 2 (erosion). In five of been attacked by the test organisms; 4) de- the wood-degrading fungi, all of which gave termination of the weight losses in various rise to attacks of Type 1, none of the cellulose substrates during culture in nutri- enzymes mentioned could be found. Only ent solutions and 5) determination of the one or two of the enzymes could be noted in occurrence of cellulase and xylanase in the remainder of the wood-degrading fungi. culture filtrates from cultures on cellulose Among the fungi which lacked the ability to substrates in liquid solutions. degrade birch wood, xylanase, could be found in connection with one species while The influence of various factors on the cellulase and mannanase could not be dis- cellulase activity of the fungi was studied. covered in relation to either of these fungi. The fact that one or more of the three cell 5) a cell-bound enzyme. Possible explana- wall degrading enzymes could not be detec- tions may be 1) that the production of ted in all of the wood-degrading fungi is cellulase is induced by substances such as considered to be a result of deficiencies in hemicellulase and lignin, which are associat- the test methods employed and not to the ed with the cellulose in wood; 2) that the inability of the fungi to produce the enzy- production of cellulase is specifically regu- me. lated by the physical structure of the wood; 3) that the cultivation conditions employed Twelve of the wood-degrading fungi appear- are unsuitable for the production of cellu- ed to be totally unable to degrade pure lase. cellulose substrate under the conditions pre- vailing in the tests described. All of these A compilation of the results obtained by the fungi caused attacks of Type 1 in birch various test methods is shown in Table 10. wood. This attack was the only expression The advantages and disadvantages of the of cellulase activity which could be demon- different methods for the determination of strated. These fungi were therefore called cellulase acitivity in microfungi are discuss- "non-cellulolytic" soft rot fungi. The fungi ed. Among other things, it is shown that could not degrade any of the pure cellulose growth on a cellulose agar medium is an substrates which were tested in agar media uncertain criterium for cellulase activity. or in liquid cultures. Some of the species The method employing the Rautela-Cowling were cultivated on birch wood meal in liquid technique appears to be superior to the cultures but no degrading of this substrate other methods. was obtained. The degradation of cotton and jute fibres in liquid cultures was studied in a The present study shows that it is not smaller test. It proved that one of the possible to prove the existance of cellulase in "non-cellulolytic" soft rot fungi, Ceratocys- certain species of cellulolytic microfungi by tis albida, achieved a significant degradation means of several generally employed me- of jute fibres while cotton was not attacked. thods. In studies of a group of microfungi by The fungus formed soft rot cavities in the means of conventional methods it may jute fibres but evidently lacked the ability to therefore be anticipated that a number of accomplish this in cotton fibres. cellulolytic species will not be detected. This work proposes a procedure, consisting of During cultivation on a pure cellulose sub- two methods, for the determination of strate in a liquid culture, small amounts of cellulase activity in microfungi. The ability glucose (as "start-glucose") had no stimula- of the fungi to produce clearing zones in ting effect on the cellulase production of the cellulose agar is studied first with the Raute- "non-cellulolytic" soft rot fungi. la-Cowling technique. All species which do not show cellulolytic activity with this meth- Various explanations of the anomalous be- od are then tested on birch wood for the havior of "non-cellulolytic" soft rot fungi ability to form soft rot cavities. Since the are discussed. The results obtained demon- wood cellulose is degraded during the forma- strate that their inability of degrade pure tion of cavities, all species which are able to cellulose substrate is probably not due to form soft rot cavities in the birch wood must 1) weak cellulolytic activity, 2) tempera- be regarded as cellulolytic. The mentioned ture factors, 3) the lack of growth factors, procedure significantly increases the possibi- 4) the requirement of "start-glucose" or lities of discovering cellulolytic species. Acknowledgements

The present study was carried out at the King at the Biodeterioration Information Department of Forest Products, Royal Colle- Centre in Birmingham, Dr. Nigel Poole at the ge of Forestry, Stockholm. School of Agriculture in Aberdeen and Dr. K.-E. Eriksson and Mr. Paul Ander at the I wish to express my warm gratitude to Swedish Forest Products Research Labora- Professor Per Nylinder and Professor Bjorn tory. Thanks are also due to Mr. Bert Henningsson for their valuable support and Pettersson at the Swedish Forest Products encouragement in the course of this investi- Research Laboratory for the supply of a gation. I also wish to thank Professor Aino cellulase preparation. Kaarik and Dr. Hans Lundstrom for stimula- ting discussions and valuable advice. The excellent technical assistance of Ylva Andersson and Inger Nordh is greatly appre- For the supply of various substrates, I wish ciated. to thank Dr. H.O.W. Eggins and Mr. Bernard Sammanfattning

Produktionen av enzymerna cellulas, xyla- eller stationara kulturer och olika inkube- nas, mannanas och amylas har studerats hos ringstemperaturer. Resultaten visade att od- trettiosex olika arter av vedangripande mik- lingsmediets sammansattning var av avgoran- rosvampar. Svamparnas vednedbrytande for- de betydelse for vissa svampar. Vid odling pi miga var tidigare kand. Tre av arterna cellulosaagarmedia hade smi mangder av saknade formiga att bryta ned bjorkved jastextrakt en hammande effekt pi cellulas- medan de ovriga fororsakade foljande tre aktiviteten. Denna effekt var ej markbar vid angreppsmonster i bjorkved: 1) soft rot odling pi cellulosasubstrat i narlosningar. De (mogelrota) kaviteter i de sekundara cell- storsta klarningszonerna erholls pi Walseth vaggarna (= angrepp av Typ 1); 2) erosion av cellulosa och s k "ball-milled" cellulosa. Be- cellvaggarna fororsakade av svamphyfer i tydligt mindre klarningszoner erholls pi Avi- cellumina (= angrepp av Typ 2); och 3) sam- cel och saltsyrabehandlad cellulosa. De olika tidigt angrepp av Typ 1 och Typ 2. svamparterna reagerade individuellt vid od- ling i skak- eller stationar kultur. Nigra Det forutsattes a priori att de vednedbrytan- arter fororsakade de hogsta viktsforlusterna de mikrosvamparna bildade samtliga av de vid odling i skakkultur medan andra uppvisa- tre cellvaggsnedbrytande enzymerna, cellu- de storre aktivitet vid odling i stationara las, xylanas och mannanas. Nigra absoluta kulturer. mitt pi de olika enzymaktiviteterna efter- stravades inte. Undersokningarna inriktades Samtliga undersokta svampar, med undantag darfor pi att forsoka pivisa narvaron av de av Phialophora verrucosa, kunde bryta ned olika enzymerna. starkelse. Bland de vednedbrytande svampar- na kunde samtliga av de tre cellvaggsned- Svamparnas enzymaktivitet studerades med brytande enzymerna, cellulas, xylanas och foljande metoder: 1) Matning av klarnings- mannanas pivisas hos tjugo arter. Fyra av de zoner i olika substrat under svampkulturer tjugo arterna fororsakade angrepp endast av vaxande pi agarpelare i provror, Rautela- Typ 1 (kaviteter), medan ovriga fororsakade Cowling teknik (cellulas-, xylanas-, manna- nigot angrepp av Typ 2 (erosion). Hos fem nas- och amylsaktivitet), 2) tillvaxt, bildning vednedbrytande svampar, vilka samtliga for- av klarningszoner och produktion av cellulas orsakade angrepp av Typ 1, kunde ingetdera vid odling av svamparna pi cellulosa-agar- av de namnda enzymerna pivisas. Hos resten plattor, 3) bestamning av forekomsten av av de vednedbrytande svamparna kunde en- cellulas och xylanas i bjorkved som angripits dast ett eller tvi av enzymerna pivisas. Bland av testorganismerna, 4) bestamning av vikts- de svampar som saknade formiga att bryta forluster hos olika cellulosasubstrat vid od- ned bjorkved, kunde xylanas pivisas hos en ling i narlosningar, och 5) bestamning av art, medan cellulas och mannanas inte kunde forekomsten av cellulas och xylanas i kultur- pivisas hos nigondera av dessa svampar. filtrat frin odlingar pi cellulosasubstrat i narlosningar. Det forhillandet att ett eller flera av de tre cellvaggsnedbrytande enzymerna inte kunde Inverkan av olika faktorer pi svamparnas pivisas hos samtliga vednedbrytande svam- cellulasaktivitet undersoktes. Bland annat par, anses bero pi bristfalligheter i de anvan- studerades effekten av sammansattningen av da testmetoderna och inte pi att svamparna olika media, tillsatser av smi mangder glukos saknar formiga att producera enzymen. (som "start-glukos"), typ av cellulosasub- strat, olikartade odlingsbetingelser som skak- Tolv av de vednedbrytande svamparna tyck- tes helt oforrnogna att bryta ned rena ningen specifikt regleras av vedens fysikalis- cellulosasubstrat under de betingelser som ka struktur, eller 3) att de odlingsbetingelser ridde i de beskrivna forsoken. Sarntliga dessa sorn anvants ar olampliga for produktion av svampar fororsakade angrepp av Typ 1 i cellulas. bjorkved. Detta angrepp var den enda yttring av cellulasaktivitet sorn kunde pivisas. Dessa En sammanstallning av de resultat sorn er- svarnpar kallades darfor "icke-cellulolytiska" hillits rned de olika testmetoderna ges i soft rot svampar. Svamparna kunde ej bryta Tabell 10. For- och nackdelar rned de olika ned nigon av de rena cellulosasubstraten metoderna for att bestamma cellulasaktivitet som provades i agarmedia eller i narlosning- hos mikrosvarnpar diskuteras. Bland annat ar. Nigra arter odlades pi rnald bjorkved i visas att tillvaxt pi ett cellulosaagarmedium narlosningar men nigon nedbrytning av det- ar ett osakert kriteriurn pi cellulasaktivitet. ta substrat erholls ej. I ett mindre forsok Den metod dar Rautela-Cowling tekniken jarnfordes nedbrytningen av bornull och jute- utnyttjas frarnst5r som overlagsen de andra fibrer vid odling i narlosningar. Det visade sig rnetoderna. att en av de "icke-cellulolytiska" soft rot svamparna, Ceratocystis albida, istadkom en Foreliggande arbete visar, att man rned flera kraftig nedbrytning av jutefibrerna rnedan allrnant anvanda rnetoder for bestamning av bomullen ej angreps. Svampen bildade soft cellulasaktivitet hos svarnpar, inte kan pivisa rot kvaiteter i jutefibrerna men saknade cellulas hos vissa arter av cellulolytiska rnik- tydligen formiga att gora detta i bomulls- rosvarnpar. Vid undersokningar av en grupp fibrerna. Vid odling pi ett rent cellulosa- rnikrosvarnpar rned konventionella metoder, substrat i narlosning, hade smb mangder kan det darfor befaras att ett antal celluloly- glukos (sorn "start-glukos") ingen stirnule- tiska arter undgir att upptackas. I detta rande effekt pi cellulasproduktionen hos arbete foreslis ett forfarande, bestiende av dessa svampar. tvi olika metoder, for bestamning av cellu- lasaktiviteten hos mikrosvampar. Svarnpar- Olika forklaringar till fenomenet rned de nas forrniga att istadkomrna klarningszoner "icke-cellulolytiska" soft rot svamparna dis- i cellulosaagar studeras forst rned Rautela- kuteras. De resultat som erhillits visar att Cowling tekniken. De svampar, sorn ej upp- deras oformiga att bryta ned rena cellulosa- visar nigon aktivitet rned denna rnetod, substrat troligen inte beror pi 1) svag cellu- undersokes sedan rned avseende pi formigan lasaktivitet, 2) ternperaturfaktorer, 3) av- att bilda soft rot kaviteter i bjorkved. Efter- saknad av tillvaxtfaktorer, 4) krav pi "start sorn vedens cellulosa brytes ned vid bildan- -glukos" eller 5) cellbundna enzym. Mojliga det av dessa kaviteter, miste alla arter som forklaringar kan vara 1) att cellulasproduk- har formiga att bilda kaviteter i bjorkved tionen induceras av substanser sisom herni- betraktas som cellulolytiska. Detta forfaran- cellulosa och lignin, vilka ar associerade de okar avsevart rnojligheterna att upptacka rned cellulosan i ved, 2) att cellulasbild- cellulolytiska arter. References

Ahlgren, E. and Eriksson, K.-E. 1967 : Characteriza- Eriksson, K.-E. 1969: New methods for the investi- tion of cellulases and related enzymes by gation of cellulases. In Cellulases and Their isoelectric focusing, gel filtration and zone elec- Applications. Advances in Chemistry Series No. trophoresis. I1 Studies on Stereum sanguino- 95. American Chemical Society, pp. 83-104. lentum, Fomes annosus and Chrysosporium Eriksson, K.-E. and Goodell, E.W.: Pleiotropic liznorum enzymes. Acta Chem. Scand. 21, mutants of the wood-rotting fungus Polyporus 1193-1200. - adustus lacking cellulase, mannanase and xyla- Bailev. I.W. and Vestal. M.R. 1937: The significan- .T nase. (In press). ce of certain wood-destroying fungi in the study Fuller, D.B. 1970: Cellulolytic enzymes of some of the enzymatic hydr61y& of cellulose. J. wood attacking fungi. Ph. D. Thesis, University Arnold Arbor. 18, 196-205. of London. Baker, F. 1939: Role of fungi and actinomycetes in Gascoigne, J.A. and Gascoigne, M.M. 1960: The the decomposition of cellulose. Nature (Lond.) xylanases of Fusarium roseum. J. Gen. Micro- 143,522-523. biol. 22, 242-248. Basu, S.N. and Ghose, S.N. 1960: The production Henningsson, B., Henningsson, M. and Nilsson, T. of cellulase by fungi on mixed cellulosic sub- 1972: Defibration of wood by use of a white- strates. Can. J. Microbiol. 6, 265-282. rot fungus. Research Note R78, Dep. of Forest Bemiller, J.N., Tegtmeier, D.O. and Pappelis, A.J. Prod., Royal College of Forestry, Stockholm. 1969: Constitutive cellulolytic enzymes of Dip- Hirsch, H.M. 1954: Temperature-dependent cellu- lodia zeae. In Cellulases and Their Applications. lase production by Neurospora crassa and its Advances in Chemistry Series. No. 95. Ameri- ecological implications. Experientia 10 (4), can Chemical Society, pp. 188-195. 180-182. Bergman, 0. and Nilsson, T. 1967: Studier over King, B. and Eggins, H.O.W. 1973: Decay mecha- utomhuslagring av aspvedsflis vid Hornefors nisms of microfungi which might produce an sulfitfabrik. (On outside storage of aspen chips enhanced permeability in wood. Int. Biodetn. at Hornefors' Sulphite Mill). Research Note Bull. 9 (1-2), 35-43. R55, Dep. of Forest Prod., Royal College of King, K.W. and Vessal, M.I. 1969: Enzymes of the Forestry, Stockholm. cellulase complex. In Cellulases and Their App- Bose, R.G. 1963: A modified cellulosic medium for lications. Advances in Chemistry Series No. 95. the isolation of cellulolytic fungi from infected American Chemical Society, pp. 7-25. materials and soils. Nature (Lond.) 198, Kaarik, A. 1960: Growth and sporulation of 505-506. Ophiostoma. Symb. Bot. Upsal. 16: 3. Bravery, A.F. 1968: Microbiological breakdown of Levi, M.P. 1964: The fungal degradation of wood. cellulose in the presence of alternative carbon J. Inst. Wood Sci. No. 12, 56-66. sources. J. Sci. Food Agric. 19, 133-135. Levi, M.P. and Preston, R.D. 1965: A chemical and Chakravarty, T., Bose, R.G. and Basu, S.N. 1962: microscopic examination of the action of the Fungi growing on jute fabrics deteriorating soft-rot fungus Chaetomium globosum on under weather exposure and in storage. Appl. beechwood (Fagus sylv.) Holzforschung 19 (6), Microbiol. 10, 441-447. 183-190. Cowling, E.B. and Brown, W. 1969: Structural Lundstrom, H. 1973: Studies on the wood-decay- features of cellulosic materials in relation to ing capacity of the soft rot fungi Allescheria enzymatic hydrolysis. In Cellulases and Their terrestris, Phialophora (Margarinomyces) luteo- Applications. Advances in Chemistry Series. viridis and Phialophora richardsiae. Research No. 95. American Chemical Society, pp. 152- Note R87, Dep. of Forest Prod., Royal College 186. of Forestry, Stockholm. 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(Lond.) 4, 153-154. cellulolytic activity of microfungi. Stud, for. Schaefer, A.C. 1957: uber die Bildung und Wirkung suec. 104. eines zellulosespaltenden Fermentes aus Schim- Nilsson, T. 1974: Formation of soft rot cavities in melpilzen. Ber. schweiz. bot. Ges., 67, various cellulose fibres by Humicola alopallo- 218-270. nella Meyers & Moore. Stud. for. suec. 112. Seifert, K. 1966: Die chemische Veranderung der Norkrans, B. 1950: Influence of cellulolytic en- Buchenholz-Zellwand durch Moderf'aule (Chae- zymes from hymenomycetes on cellulose prepa- tomium globosum Kunze). Holz Roh- u. Werk- rations of different crystallinity. Physiol. Plant. stoff, 24, 185-189. 3, 75-87. Simpson, M.E. and Marsh, P.B. 1964: Cellulose Norkrans, B. and Aschan, K. 1953: A study in decomposition by the Aspergil1i.U.S.D.A. Ag- the cellulolytic variation for wildtypes and ric. Res. Sew. Technical Bullentin. No 1303. mutants of Collybia velutipes. 11. Relations to Smensen, H. 1952: On the specificity and products different nutrient media. Physiol. Plant. 6, of action of xylanase from Chaetomium globo- 829-836. sum Kunze. Physiol. Plant. 5, 183-198. Parbery, D.G. 1969: The natural occurrence of Sorensen, H. 1957: Microbial decomposition of Cladosporium resinae. Trans. Brit. mycol. Soc. xylan. Acta Agric. Scand. Supplement 1, 1-86. 53, 15-23. Stone, J.E., Scallan, A.M., Donefer, E. and Ahl- Park, D. 1973: A modified medium for isolation gren, E. 1969: Digestibility as a simple function and enumeration of cellulose-decomposing of a molecule of similar size to a cellulase fungi. Trans. Brit. mycol. Soc. 60 (I), enzyme. In Cellulases and Their Applications. 148-151. Advances in Chemistry Series No. 95. American Poole, NJ. and Taylor, D.E. 1973: Degradation of Chemical Society, pp. 219-238. plant fibres. Report from Division of Agricultu- Stranks, D.W. and Bieniada, J. 1971: A rapid test ral Bacteriology, Department of Agriculture, for cellulolytic activity. Int. Biodetn. Bull. 7 University of Aberdeen. (31, 109-111. Rautela, G.S. and Cowling, E.B. 1966: Simple Traaen, A.E. 1914: Untersuchungen iiber Boden- cultural test for relative cellulolytic activity of pilze aus Nonvegen. Nytt Mag. Naturvidensk. fungi. Appl. Microbiol. 14 (6), 892-898. 52,20-121. Reese, E.T. and Levinson, H.S. 1952: A compara- Walseth, C.S. 1952: Occurrence of cellulases in tive study of the breakdown of cellulose by enzyme preparations from microorganisms. microorganisms. Physiol. Plant. 5, 345-366. Tappi 35, 228-233. Reese, E.T., Levinson, H.S., Downing, M.H. and Wilhelmsen, G. 1965 : Mikromorfologiske og bio- White, W.L. 1950: Quartermaster culture col- kjemiske forandringer i trevirke ved enzymatisk lection. Farlowia 4 (I), 45-86. nedbrytning. Norsk Skogsind. 19, 187-193. 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Table 1. List of organisms Strain Acremonium atro-griseum (Panasenko) W. Gams SP35-7 (=CBS 981.70) Bispora betulina (Corda) Hughes P175-26 Catenularia heimii Mangenot CBS 141.53 Ceratocystis albida (Mathiesen-Kaarik) Hunt B-23 Ceratocystis olivacea (Mathiesen) Hunt B-57 Ceratocystis stenoceras (Robak) C. Moreau B-I04 Chrysosporium pannorum (Link) Hughes SP47-16 Cladorrhinum sp. A 600-2 Cladosporium resinae (Lindau) de Vries 13385 Cladosporium sp. A SP78-4 Coniothyrium fuckelii Sacc. var. sporulosum W. Gams & Domsch CBS 218.68 Cordana pauciseptata Preuss B63-A-25 Dictyosporium elegans Corda SP5-16 (= CBS 946.70) Gonatobotrys sp. A SP37-6 Graphium sp. A B68-A-16 Humicola alopallonella Meyers & Moore CBS 207.60 Humicola grisea Traaen SP37-22 Mollisia sp. A T694C Petriellidium boydii (Shear) Malloch SP3 1-4 Phialocephala dirnorphospora Kendrick CBS 300.62 Phialocephala sp. A P152-55 (=CBS 390.71) Phialocephala sp. C 1) 71257-13 Phialophora fastigiata (Lagerb. & Melin) Conant 731-1-3b Phialophora hoffmannii (van Beyma) Schol-Schwarz SP33-4 Phialophora verrucosa Medlar P152-8 (= CBS 839.69) Phialophora sp. A SP35-1 (= CBS 882.73) Pseudeurotium zonatum van Beyma SP17-2 Rhinocladiella anceps (Sacc. & Ellis) Hughes SP35-15 Rhinocladiella sp. A P160-14 Scytalidium lignicola Pesante H97 - 1 Scytalidium sp. B 721009-2 Wardomyces inflatus (March.) Hennebert P180-62 (= CBS 412.68) Xylogone sphaerospora v. Arx & Nilsson E7-2 (= CBS 186.69) Fungus A A40-1 Fungus B SP3-10 Fungus D B77-A-23

Strain number within parenthesis refer to species which have been included in the culture collection at "Centraalbureau voor Schirnmel-cultures" in Baarn.

1) Recently identified as Phialocephala fusca Kendrick. Table 2. Depth of clearing (mm) of Walseth cellulose, ball-milled cellulose, Avicel, HC1-treated cellulose, larch xylan, pine glucomannan and starch in agar columns (RautelaCowling technique).

Species Type of attack Walseth cellulose Ball-milled cellu- on birch wood 1) lose R-C medium B-VII medium B-VII medium 21 days 42 days 21 days 42 days 21 days 42 days Acremonium atro-griseum Bispora betulina Catenularia heimii Ceratocystis albida Ceratocystis olivacea Ceratocystis stenoceras Chrysosporium pannorum Cladorrhinum sp. A Cladosporium resinae Cladosporium sp. A Coniothyrium fuckelii var. sporulosum Cordana pauciseptata Dictyosporium elegans Gonatobotrys sp. A Graphium sp. A Humicola alopallonella Humicola grisea Mollisia sp. A Petriellidium boydii Phialocephala dimorphospo~ Phialocephala sp. A Phialocephala sp. C Phialophora fastigiata Phialophora hoffmannii Phialophora verrucosa Phialophora sp. A Pseudeurotium zonatum Rhinocladiella anceps Rhinocladiella sp. A Scytalidium lignicola Scytalidium sp. B Wardomyces inflatus Xylogone sphaerospora Fungus A Fungus B Fungus D - Notes 1) Data from Nilsson (1973) except for Ceratocystis albida, Ceratocystis stenoceras, Cladosporium sp. A and Fungus D. These fungi were tested for type of wood attack according to the methods described previously. 2) Cellulose powder (Whatman CF 11) treated with concentrated hydrochloric acid according to Bose (1963). Type of attack 0 no attack, 1 Type 1 attack (cavities), 2 Type 2 attack (erosion). Cellulolytic activity + definite clearing, but measurements not possible due to diffuse zonal fronts. D diffuse clearing. Substrate Avicel HC1-treated Xylan Glucomannan Stach cellulose 2) R-C medium B-VII medium B-VII medium B-VJI medium B-VII medium B-VII medium 21 days 42 days 21 days 42 days 21 days 42 days 21 days 42 days 21 days 42 days 21 days 42 days Table 3. Relative growth on two cellulose agar media, clearing of cellulose in agar plates and demonstration of cellulolytic activity by the use of P a an agar-plate diffusion technique with mycelium-agar plugs.

Clearing of cellulose in the agar Clearin of cellulose agar around transferred agar Species Time Relative growth on cellulose agar 1) test plates 2) plugs 27 (days) Medium F6A Medium B-VII Medium F6A Medium B-VII Plugs from medium F6A Plugs from medium B-VII

Acremonium atro-griseum 21 2 - + Bispora betulina 21 2 1 - - - - 42 2 1 - - - -

Catenularia heimii 21 1 1 - - - - 42 1 1 - - - - Ceratocystis albida 21 1 1 - - - - 42 1 1 - - - - Ceratocystis olivacea 21 1 1 - - - - 42 1 1 - - - -

Ceratocystis stenoceras 21 1 1 - - - - 42 1 1 - - - - Chrysosporium pannorum 21 2 - + Cladorrhinum sp. A 2 1 3 + + Cladosporium resinae 21 2 - - 42 2 - - Cladosporium sp. A 21 2 - - 42 2 - + Coniothyrium fuckelii var. sporulosum 21 3 + + Cordana pauciseptata 21 1 - + Dictyosporium elegans 21 2 - + Gonatobotrys sp. A 21 1 - - 42 1 - -

Graphium sp. A 21 1 - - 42 1 - -

Humicola alopallonella 21 2 - - 42 2 - - Humicola grisea 21 3 + +

Mollisia sp. A 2 1 2 - - 4 2 2 - -

Table 4. Cellulase and xylanase activity in birch wood blocks attacked by 30 species of the test fungi.

Species Cellulase Xylanase Agar medium (1)

Acremonium atro-griseurn Bispora betulina Catenularia heimii Ceratocystis albida Ceratocystis stenoceras Chrysosporium pannorum Cladorrhinum sp. A Cladosporium resinae Coniothyrium fuckelii var. sporulosum Cordana pauciseptata Dictyosporium elegans Gonatobotrys sp. A Graphium sp. A Hurnicola alopallonella Humicola grisea Mollisia sp. A Petriellidium boydii Phialocephala sp. A Phialophora fastigiata Phialophora hoffmannii Phialophora verrucosa Phialophora sp. A Pseudeurotium zonatum Rhinocladiella anceps Rhinocladiella sp. A Scytalidium lignicola Wardomyces inflatus Xylogone sphaerospora Fungus A Fungus B

Notes I) The agar medium used in the decay test C = Cellulose agar F6A M = 2.5 percent malt extract agar Table 5. Growth on the nutrient solutions EP and B-VII-L with added glucose

EP (Control. No nlucose added) EP+ glucose (20 g/l) B-VII-L+ glucose (20 g/l) Species Time Weight of Weight of Weight of (days) mycelium (mg) Final pH mycelium (mg) Final pH mycelium (mg) Final pH

Bispora betulina

Catcnularia heimii

Ceratocystis albida

Ceratocystis olivacea

Ceratocystis stenoceras

Cladosporium resinae

Cladosporium sp. A

Gonatobotrys sp. A

Graphium sp. A

Mollisia sp. A

Phialocephala dimorphospora g Table 5. (Continued).

EP (Control. No glucose added) EP +glucose (20 g/l) B-VII-L+ glucose (20 g/l) Species Time Weight of Weight of Weight of (days) mycelium (mg) Final pH mycelium (mg) Final pH mycelium (mg) Final pH

Phialocephala sp. A 14 97.2 2.6 21 112.4 2.6 32.0 2.5 28 120.1 2 .7 Phialocephala sp. C 14 13.2 3.7 21 20.2 4.9 44.2 2.5 28 28.4 6.2 Phialophora verrucosa 14 42.4 3.6 21 56.8 3.8 22.6 2.6 28 75.1 3.0 Phialophora sp. A 14 49.0 3.4 21 93.0 3.0 32.2 2.6 28 126.3 2.7 Rhinocladiella sp. A 14 16.5 3.1 21 28.4 2.9 43.9 2.5 28 39.2 2.8 Scytalidium sp. B 14 102.9 2.9 21 129.9 2.6 82.9 2.4 28 105.2 2.9 Fungus A 7 19.3 3.6 15.6 3 .0 14 52.9 3.5 30.4 2.7 21 84.1 3.5 62.5 2.4 2 8 122.4 3.0 94.0 2.4 Fungus B 14 29.9 3.5 21 97.9 3.4 32.6 2.7 28 107.8 3.1 Fungus D 14 47.0 3.2 21 68.4 2.9 30.4 2.5 28 96.1 2.7 Table 6. Degradation of cellulose N, Avicel and cotton, and the production of cellulase and xylanase in liquid cultures with medium EP.

Culture conditions Species Substrate Time Shake cultures Stationary cultures (days) Weieht loss (%) Cellulase Xvlanase Final DH Weieht loss (%) Cellulase Xvlanase Final DH Control (not inoculated) Avicel Cotton Cellulose N Acremonium atro-griseum Avicel Cotton Cellulose N Bispora betulina Avicel

Cotton

Cellulase N

Catenularia heimii Cellulose N

Ceratoc ystis albida Cellulose N

Ceratocystis olivacea Cellulose N

Ceratocystis stenoceras Cellulose N

Chrysosporium pannorum Avicel Cotton Cellulose N Cladorrhinum sp. A Avicel Cellulose N Cladosporium resinae Cellulose N

Cladosporium sp. A Cellulose N

V, + Coniothyrium fuckelii Avicel var. sporulosum Cellulose N Table 6. (Continued)

Culture conditions Species Substrate Time Shake cultures Stationary cultures -- -- (days) Weight loss (%) Cellulasc Xylanasc Final DH Weieht loss (%) Cellulase Xvlanase Final VB

Cordana pauciseptata Avicel Cotton Cellulose N Dictyosporium elegans Avicel

Cotton

Cellulose N

Gonatobotrys sp. A Avicel

Cotton

Cellulose

Graphium sp. A Avicel Cotton Cellulose N Humicola alopallonella Avicel

Cellulose N

Humicola grisea Avicel Cotton Cellulose N Mollisia sp. A Avicel

Cotton

Cellulose N 3Q'990909'?Q!ml'?'?9U?t'9'?'??\4 1"9'?" '?N9r? G wr-t- ww r-w r-w ww ww ww ww ww r-t- t-t- r-t- r-w

I Ill I I ++ I ++ II I II Il I1 I I

I Ill I1 I I+ ++ ++ I II I1 II 11 II ++ Table 6. (Continued)

P Culture conditions Species Substrate Time Shake cultures Stationary cultures (days) Weight loss (%) Cellulase Xylanase Final pH Weight loss (%) Cellulase Xylanase Final pH - Rhinocladiella anceps Avicel

Cotton

Cellulose N

Rhinocladiella sp. A Avicel Cotton Cellulose N

Sytalidium lignicola Avicel Cellulose N Sytalidium sp. B Cellulose N

Wardomyces inflatus Avicel Cotton Cellulose N Xylogone sphaerospora Avicel Cotton Cellulose N Fungus A Avicel

Cotton

Cellulose N

Fungus B Cellulose N

Fungus D Cellulose N Table 7. Degradation of cellulose N, Avicel and cotton, and the production of cellulase and xylanase in liquid cultures with medium B-VII-L.

Culture conditions Species Substrate Time Shake cultures Stationary cultures (days) Weight loss (%) Cellulase Xvlanase Final pH Weight loss (%) Cellulase Xylanase Final pH - Acremonium atro-griseum Avicel 21 Cotton 21 Cellulose N 21 Bispora betulina Avicel 21 4 2 Cellulose N 21 42 Catenularia heimii Cellulose N 21 42 Ceratocystis albida Cellulose N 2 1 4 2 Ceratocystis olivacea Cellulose N 2 1 42 Ceratocystis stenoceras Cellulose N 21 42 Cladosporium resinae Cellulose N 21 4 2 Cladosporium sp. A Cellulose N 21 4 2 Cordana pauciseptata Avicel 21 Cotton 21 Cellulose N 2 1 Dictyosporium elegans Avicel 21 Cotton 21 Cellulose N 21 Gonatobotrys sp. A Cellulose N 2 1 4 2 Graphium sp. A Cellulose N 2 1 4 2 vl vl Humicola alopallonella Cellulose N 2 1 4 2 Table 7. (Continued).

Culture conditions Species Substrate Time Shake cultures Stationary cultures (days) Weirht loss (%) Cellulase Xylanase Final pH Weight loss (%) Cellulase Xylanase Final pH

Humicola grisea Avicel Cotton Cellulose N Mollisia sp. A Cellulose N

Phialocephala Cellulose N dimorphospora Phialocephala sp. A Avicel

Cellulose N

Phialocephala sp. C Cellulose N

Phialophora verrucosa Cellulose N

Phialophora sp. A Avicel

Cotton

Cellulose N

Rhinocladiella anceps Avicel Cellulose N Rhinocladiella sp. A Cellulose N

Scytalidium lignicola Cellulose N Scytalidium sp. B Cellulose N Table 7. (Continued).

Culture conditions Species Substrate Time Shake cultures Stationary cultures - - (days) Wei~htloss (%) Cellulase Xylanase Final pH Weight loss (%) Cellulase Xylanase Final pH

Fungus A Avicel 2 1 4 2 Cotton 2 1 42 Cellulose N 2 1 42 Fungus B Cellulose N 21 4 2 Fungus D Cellulose N 2 1 42 Table 8. Degradation of cotton and jute fibres, and the production of cellulase and xylanase in liquid cultures with medium EP.

Cotton Jute Species Time (days) Weight loss % Cellulase Xylanase Final pH Weight loss % Cellulase Xylanase Final pH

Control (not inoculated) 4.4 4.2 4.2

Bispora betulina

Ceratocystis albida

Phialophora sp. A

Wardomyces inflatus

Xylogone sphaerospora r- 9 1 CQ * L? wt-t- morn

I I I I++

I I1 I++

"Nu! 2: 2 wwm

1" I++

I I++

-! a C? a?? r- t- t- WWd

I I I I++

I I++

2 2 2 z z 2

I I I I++

Ill + + +

2 2 2 22;

!!I I++

I I I + + +

'?L?C? YC?" t- t- t- www

I I I I++

Ill +++ t-d- t-dm - N - N

-.-a C Q D 4 m M s 2 Q I 2 Lr, Table 10. Summation of the results obtained with the different test methods.

Cellulase Xylanase Mannanase Amylase Species Mycelium- Liquid cultures Liquid cultures rest tubes agar plugs Wood blocks Weight lossl) Culture filtrate Test tubes Wood blocks Culture filtrate Test tubes Test tubes Acremonium atro-griseum Bispora betulina Catenularia heimii Ceratocystis albida Ceratocystis olivacea Ceratocystis stenoceras Chrysosporium pannorum Cladorrhinum sp. A Cladosporium resinae Cladosporium sp. A Coniothyrium fuckelii var. sporulosum Cordana pauciseptata Dictyosporium elegans Gonatobotrys sp. A Graphium sp. A Humicola alopallonella Humicola grisea Mollisia sp. A Petriellidium boydii Phialocephala dimorphospora Phialocephala sp. A Phialocephala sp. C Phialophora fastigiata Phialophora hoffmannii Phialophora verrucosa Phialophora sp. A Pseudeurotium zonatum Rhinocladiella anceps Rhinocladiella sp. A Scytalidium lignicola Table 10. (Continued).

Cellulase Xylanase Mannanase Amylase Species Mycelium- Liquid cultures Liquid cultures Test tubes agar plugs Wood blocks Weight lossl) Culture filtrate Test tubes Wood blocks Culture filtrate Test tubes Test tubes

Scytalidiurn sp. B Wardornyces inflatus Xylogone sphaerospora Fungus A Fungus B Fungus D

Positive results Negative results

Notes 1) Significant weight loss of cellulose N + Activity present - Activity absent NTNot tested 2) Only 30 species tested