RELATIVE AERATION REQUIREMENTS 

BY SOFT ROT AND BASlDlOMYCETE 

WOOD-DESTROYING FUNGI 

May 1961 

No. 2218 
- - - - - - 

RELATIVE AERATION REQUIREMENTS BY SOFT ROT 

AND BASIDIOMYCETE WOOD-DESTROYINGFUNGI 

By 

CATHERINE G. DUNCAN, Pathologist 

1 

Forest Products Laboratory, Forest Service 

U.S. Department of Agriculture 

Summary 

Isolates of 50 soft-rot fungi and 41 Basidiomycete decay fungi were studied for growth responses 

in sealed and partially closed culture tubes. All isolates maintained a uniform rate of linear 

growth until oxygen was exhausted in the tubes, which suggests that respiration was mediated 

through the cytochrome system. Linear growth was suppressed more by lack of aeration with the 

soft rotters than with the Basidiomycetes, but the reserve was true as regards many soft rotters 

isolated from cooling towers, when growth was measured by the weight of mycelium produced. 

Taking total growth (weight of mycelium) as the preferable index of relative tolerance of 

restricted aeration, it is concluded that the prevalence of soft rot in the wet wood of cooling 

towers, and the near absence of Basidiomycetes, is attributable at least in part to a superior 

tolerance of poor aeration on the part of the soft-rot fungi concerned. No significant difference 

in growth responses was noted between white rotters and brown rotters in the Basidiomycete group. 

Introduction 

Certain Ascomycetes and imperfect fungi are capable of causing rot in very wet Wood where decay 

by Basidiomycete fungi cannot take place although temperature and conditions of nutrition are 

quite adequate. One probable explanation for this difference in decay capacity is that the soft 

rotters may require less oxygen, since much of the air in the wood cells is replaced by water. 

Another conceivable explanation is that the soft rotters may have a greater tolerance for the 

increase in carbon dioxide, the appearance of exudate materials--the so-called staling products--or 

a change in pH of the surroundings. Both reproductive processes and the vegetative growth of fungi 

are known to be affected by a severely limited supply of oxygen or increased quantities of carbon 

dioxide, conditions which might be present in a poorly aerated environment. 

The present study was planned to obtain information on the rates of growth of representative fungi 

causing the two types of decay, when the fungi were grown on malt agar, over which the exchange 

of air either was limited by requiring it to take place through water or else was entirely prevented. Quantitative measurements of oxygen depletion and carbon dioxide build-up were not made. 

However, it was hoped that differences in growth might be found between the fungus groups when 

subjected to uniformly restricted aeration. Such differences would indicate physiological 

characteristics that might supply a clue as to why some Ascomycetes and imperfect fungi, such as 

those causing soft rot, are capable of attacking wood under conditions of wetness seldom tolerated 

by the Basidiomycetes. 

1Maintained at Madison, Wis., in cooperation with the University of Wisconsin. 

Report No. 2218 
Methods 

Growth of the fungi Was determined by (1) linear rates of growth and (2) total dry weight of 

mycelium produced on malt agar in modified test tubes (6)2 whose openings were closed with either 

cotton, rubber stoppers, or water of a given depth. 

The test tubes were 20 centimeters long by 2 centimeters in diameter at the mouth. They had been 

modified by a deep indentation of the wall on one side near the mouth, so as to form a dam on the 

lower side of the tube to prevent escape of the liquid when the sterilized medium was cooled and 

solidified with the tube in a horizontal position. This resulted in a uniform, narrow strip of 

substrate along one side of the tube, approximately 15 centimeters long, 2 centimeters wide, and 

about 1 centimeter deep along the middle. In order to keep the substrate in place when the tubes 

were later placed in a vertical position, the tubes were not disturbed until the strips had been 

allowed to harden for 24 hours. 

The malt agar medium contained 2 percent each of Difco malt and agar in distilled water, with the 

pH adjusted to 6 before sterilization. This nutrient was not necessarily optimum for all the fungi 

to be used hut was known to permit good, uniform growth. Fifteen cubic centimeter6 of the melted 

agar were put into each test tube, which was then plugged firmly with cotton and autoclaved at 

15 pounds of pressure (121° C.) for 15 minutes. 

The inoculum (approximately a 3-millimeter cube) was cut with a two-pronged blade from the growing 

margin Of a Petri-dish culture so as to provide mycelium 2 to 7 days old. The inoculum was placed 

at the forward end of the agar strip, mycelial surface downward, so that new growth started 

directly on the medium and extended linearly toward the closed end of the tube. For most fungi, 

the advancing margin of new growth was nearly straight and at right angles to the long axis of a 

tube. Six tubes were inoculated with each fungus isolate. 

The inoculated tubes were placed in an incubation room on racks that held them vertically, with 

the rounded ends uppermost. This position of the tube was necessary in order to keep the maltagar surface free of condensed water, which prevented an even growth margin. 

The incubation room was maintained at 80° F. (26.7° C.) and 70 percent relative humidity. Although 

this temperature was optimum for most of the Basidiomycetes, it was aomewhat below that for many 

of the imperfect fungi studied (3). 

Measuring Linear Growth 

As soon as the fungus started to grow from the inoculum onto the agar strip, the measurements of 

linear growth were begun by marking on the culture tube the position of the advancing mycelial 

margin, using for the purpose a ground-glass marking line made on the same side of the tube as the 

substrate. This mark was considered the starting point for the growth record, and was made at 

the same time for all six tubes inoculated with a particular isolate. 

Immediately after the initial marking, the cotton plugs were removed from of the 6 tubes containing a given fungus and replaced with two different types of closure, each intended to limit 

aeration of the cultures. A rubber stopper was inserted tightly into two of the four tubes 

(subsequently referred to as "sealed" tubes) and the connection covered with wax to prevent air 

exchange. Water was placed in the open necks of the other two tubes (subsequently referred to 

as "water-closed tubes") to a distance of about 1 inch with the purpose of limiting but not preventing air exchange. This type of closure was accomplished by placing the tubes upside down 

in wooden racks so that the mouths extended 1 inch below the water surface. The water was 

maintained at a depth of 2 inches in a large pan. Observations of growth were mode without 

removing the tubes from the water. The remaining two tubes inoculated with a given isolate 

(socalled "normal" tubes) Were left, with the cotton plugs, through which exchange of air could 

take place relatively freely. All the tubes were incubated for the duration of the growth studies 

in a room maintained at 80° F. (26.7° C.) and 70 percent relative humidity. 

2Underlined numbers in parentheses refer to the literature cited at the end of this report. 

Report No. 2218 -2-
Growth of the fungi was measured every 24 hours by viewing the margin of the mycelium through the 

substrate and marking it on the ground glass strip. Observations Were made of the two sealed 

and corresponding two normal tubes until 48 hours after linear growth had ceased in the former. 

This meant that the record of linear growth for the normal tubes covered a period 48 hours longer 

than that for the sealed. Linear growth in the two water-closed tubes likewise was recorded until 

it ceased. 

At the completion of the linear growth observations, the distances between the markings on the 

tubes, representing the daily growth rates, were measured to the nearest millimeter, and the 

average daily rates with the respective tube closures were computed from these measurements. 

Measuring Growth by Dry Weights 

After the completion of the linear growth studies, the dry weight of the mycelium produced in 

each sealed and normal tube was determined. The growth tubes were opened, filled to within 1 inch 

of the top with water, and steamed for 30 minutes to separate the mycelial mat from the agar. 

The melted contents of a tube were then poured through a tared filter paper, and the mycelial 

residue was washed by passing approximately 250 cubic centimeters of boiling water through the 

filter system. The filter paper and mycelium were then oven-dried and weighed, and the weight of 

the mycelium was computed. 

Selection of Fungi 

A list of the 91 fungi used in these growth studies is found in table 1. Of this number, 41 were 

Basldiomycetes, including 21 white and 20 brown rotters. None of these Basidiomycetes, except 

Poria nigrescens and Peniophora mollis, are known to cause frequent decay in wood that is consistently very wet. These two white-rot exceptions cause considerable decay in the structural 

members, and occasionally in slats of water cooling towers. 

Fifty of the 91 isolates were soft-rot fungi, 47 being identified as Fungi Imperfecti and three as 

Ascomycetes. All had been isolated either from wood in cooling towers or from wood in contact 

with soil. Four genera--Acremonium, Cephalosporium, Phoma, and Phialophora--were represented by 

mare than one isolate, since they are most frequently isolated from cooling tower slats. Likewise, members of these four genera have shown considerable capacity to produce soft rot under 

laborntory conditions. 

Results 

Before examining the differences in growth results among the fungi, it is of interest to note a 

characteristic of their growth under reduced aeration which indicated a particular type of 

respiration. From plottings of the amount of growth against length of time (fig. 1), it was 

evident that the rate of linear growth by a given fungus did not vary significantly from the 

starting point until growth ceased because of lack of oxygen. This straight-line relation of 

growth to time over the entire period of development was maintained, without observed exception, 

by both soft-rot and Basidiomycete fungi grown in either the sealed or water-closed tubes. 

In plants and animals, the type of respiration that proceeds at a regular rate until the oxygen 

supply is essentially exhausted is commonly associated with the cytochrome system. According 

to Cochrane (2), the cytochrome system appears to be general in fungi as judged by spectroscopic 

observations, although such measurements cannot indicate with precision whether the components 

of a new system are the same as those of the more familiar yeast and animal system. To check 

this general assumption, a soft rotter (Acremonium sp., R48B) and a brown rotter (Poria monticola, 

Mad., 698) were examined visually with a microspectroscope3 after the heavy mycelial suspensions

had been treated with sodium hydrosulfite (Na2S2O4) to reduce the cytochromes. The appearance 

of bands near 552, 562, and 605 millimicrons indicated the presence of cytochromes c, b, and a. 

3The possibility that the cytochrome system might be involved here was suggested by R. H. Burris 

of the University of Wisconsin. Dr. Burris also made the microspectroscope observations. 

Report No. 2218 -3-
Linear Growth Studies in the Sealed 

and Water-Closed Tubes 

A summary of the linear growth rates relative to the normal, and of the total number of days over 

which growth could take place in the sealed and water-closed tubes, is given in table 2 for the 

50 soft-rot fungi and the 41 brown- and white-rot Basidiomycetes. These data indicate that, for 

the majority of fungi, differences in linear growth between cultures in the water-closed and 

sealed tubes Were small, being for all types slightly greater in the water-closed than in the 

sealed. Similar small differences were exhibited in the number of days through which the fungi 

were able to grow in the two types of tubes. These small differences were disappointing, since it 

had been hoped that the water-closed tubes would be less restrictive of growth and, therefore, 

would provide comparisons among the fungi at growth-retarding but not inhibiting levels of aeration. 

It seems noteworthy in this respect only that the slight superiority of development in the waterclosed tubes was consistently greater in the case of the Basidiomycetes than with the soft rotters. 

One striking difference in linear growth unrelated to the type of culture tube is brought out by 

table 2. The percentage of normal linear growth by the Basidiomycetes (95 percent) was much 

greater than that by the soft rotters (67 percent). There was no important difference in relative 

growth rates between the white rotters and brown rotters. 

The marked difference in relative growth rates between the soft-rot and Basidiomycete fungi in 

sealed tubes is additionally apparent from a comparison of the frequency distribution of the fungi 

in various growth rate groups. Such a comparison is provided by the bar graphs in figure 2. 

Included in the figure are separate data for four genera of soft rotters that have been most 

frequently isolated from cooling towers. It may be noted that, all the brown rotters and most of 

the white rot isolates maintained a linear growth rate that was at least 80 percent of normal. 

By contrast, only about one-fourth of the 50 soft rot isolates attained as high a relative growth 

rate. Without additional data, it cannot be said how typical may be the moderate differences shown 

in figure 2 between all the soft rot isolates and the four selected soft, rot genera, or between the 

white rot and brown rot species. In any case, the respective differences are not indicated to be 

large enough to be of practical importance. 

It was expected, of course, that fungi which normally inhabit the wettest and most poorly areated 

wood Would be more capable than others of approaching normal growth rates in the sealed tubes. 

However, the growth rate of the cooling tower isolates in the genera, Acremonium, Cephalosporium, 

Phialophora, and Phoma, were no less affected by the closed tube condition than the soft rotters 

in general. Thus, neither in this comparison within the group of soft-rot fungi, nor in the 

comparison of relative growth rates of the soft rotters and the Basidiomycetes in sealed tubes, 

is there a clue as to why soft rotters appear to he more tolerant of very wet, poorly aerated wood 

than are brown- or white-rot fungi. Such a clue was provided, however, by comparisons of total 

production of mycelium--as contrasted with amounts evidenced only by linear growth. 

Comparison of Growth Capacities Measured by 

the Dry Weight of Mycelium Produced 

The dry weight of the mycelium of each fungus isolate in the sealed and normal tubes was obtained 

as soon as linear growth ceased in the sealed tubes. The mean dry weights of mycelium, as 

percentages of normal weight, are shown in table 3. The mean mycelial weights of the brown-, 

White-, and soft-rot fungi in the sealed tubes were 78, 81, and 87 percent, respectively, Of the 

normal weights. The four genera of soft rotters most frequently isolated from cooling towers 

exhibited on the average no decrease in rate of mycelium production (mean growth percent equals 

104). Thus, although these figures indicate no marked difference in mycelium production under 

restricted aeration between the Basidiomycetes and the soft rotters as entire groups, the 

difference is the opposite of that observed for linear growth. It seems particularly significant, 

moreover, that the four principal cooling-tower isolates were able to maintain fully their normal 

rate of growth until the oxygen supply was exhausted. 

A superior capacity of these cooling-tower fungi to develop with limited oxygen is more strikingly 

evidenced in the growth-frequency diagrams of figure 3. Approximately 80 percent. of the soft rot 

isolates, representing Acremonium, Cephalosporium, Phialophora, and Phoma species, produced a 

weight of mycelium amounting to not less than 90 percent. of normal, in contrast to no more than 

about 50 percent of the Basidiomycete species. 

Report No. 2218 -4-
No significant difference between the white- and brown-rot fungi in capacity to maintain production 

of mycelial mass is indicated. 

Discussion and Conclusions 

Measurement of growth by the linear development of the mycelium on malt-agar indicated that the 

brown- and white-rot fungi (Basidiomycetes) were capable of greater growth than the soft-rot fungi 

where air was excluded from the culture tube. Measurement of growth by the dry weight of the 

mycelium produced indicated that the soft-rot fungi most frequently isolated from the wet wood of 

cooling towers were capable of greatest growth under such conditions. 

Since dry weight reflects the total rather than a part of the development of a fungus, and for 

other reasons that will follow, weight rather than linear growth of mycelium is believed to be the 

better index of the tolerance of restricted aeration by these fungi. From this standpoint, the 

isolates in the four genera of soft-rot fungi most frequently isolated from cooling 'towers exhibited 

superior tolerance. This furnishes at least a partial explanation of why soft rot, but seldom 

brown or white rot, is found in the wetter wood of cooling towers. It seems pertinent to add that 

Basidiomycete decay fungi typically do not invade wood that is wet enough to materially limit the 

ingress of oxygen. 

The reason for the differences between linear and weight growth of the fungi under the conditions 

of poor aeration is not known. Other investigators have found, however, that these two measurements 

of growth can differ considerably, and it has been concluded by some that mycelial extension 

sometimes is a poor criterion of development. In comparing the diameters and dry weights of 

similarly aged Petri-dish cultures of Ceratostomella ulmi in the presence of varying amounts of 

pyridoxine, Fries (4) found that the average size of 5-day-old colonies without pyridoxine Was 

16.3 millimeters, whereas that of colonies receiving pyridoxine was 12.3 millimeters. The weights 

of mycelium produced under these two conditione were in the reverse order--5.2 and 18.1 milligrams, 

respectively. Similar differences between diameters of growth and dry weights of C. fimbriata 

also have been shown by Lilly and Barnett (5). 

Brancato and Golding (1), on the other hand, came to the conclusion that the diameter of a fungus 

colony is sufficiently reliable for determining growth rates because there is no acceleration of 

growth rate with time under a given set of conditions. They did not, however, directly compare 

colony diameters with weight except when determining the effects of depths of media. In their 

comparison, Aspergillus niger, A. flavus, and Penicillum notatum produced the same diameter of 

colonies on media 1 or 4 millimeters in depth, hut the weight of mycelium was 2 to 30 milligrams 

greater on media with the greater depth. 

In the present study, maintenance of a normal weight production of mycelium by the four genera of 

cooling-tower fungi despite a reduced rate of linear growth probably has a simple explanation. It 

is assumed--with limited confirmatory observations--that the mycelium was developed by these 

fungi at a greater than usual depth in the agar substrate, in amounts sufficient to offset the 

lesser surface development. It would follow, of course, that those Basidiomycetes which were 

able to maintain a normal rate of linear growth, did so at the expense of their usual depth Of 

subsurface development. 

Report No. 2218 -5-
Literature Cited 

(1) Brancato, F. P., and Golding, N. S. 

1953. The Diameter of the Mold Colony as a Reliable Measure of Growth. Mycologia 45:848­

864. 

(2) Cochrane, V. W. 

1958. Physiology of Fungi. John Wiley and Sons, Inc., New York 

(3) Duncan, C. G. 

1960. Wood-Attacking Capacities and Physiology of Soft-Rot Fungi. Forest Products 

Laboratory Report No. 2173. 

(4) Fries, N, 

1943. Die Einwirkung von Adermin Aneurin und Biotin auf das Wachstum einiger Ascomyceten. 

Symbolae Botan. Upsaliensis 7:1-73. 

(5) Lilly, V. G., and Barnett, H. L. 

1951. Physiology of the Fungi. McGraw-Hill Book Company, New York. 

(6) Scheffer, T. C. 

1935. A Tube for Culturing Fungi. Science 82:467-468. 

2.-13 Report No. 2218 -6-
Table 1.--Soft rot and Basidiomycete fungi whose relative aeration 

requirements were assessed in normal, sealed, and waterclosed tubes 

Report No. 2218 
Table 2.--Relative rates of linear growth and the number of days 

until stoppage of growth by the different types of 

fungi in sealed and water-closed tubes 

Table 3.--Relative amounts of mycelium produced by the different 

types of fungi In sealed tubes 

1Isolates in genera Acremonium, Cephalosporium, Phialophora, and Phoma. 

Report No. 2218 
Figure 1. --Examples of linear growth-time relations observed for white-, brown-, and soit-rot 

fungi growing on malt agar in normal, sealed and water-closed tubes. 
Figure 2. --Frequency distribution of the fungi according to indicated 

relative capacities for linear growth in sealed tubes. 

Report No. 2218 
Figure 3. --Frequency distribution of the fungi according to indicated 

relative capacities for increasing mycelial weight in the sealed 

tubes. 

Report No. 2218 
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