Soil Bid. Biochem. Vol. 5, pp. 505-515. Pergamon Press 1973.

Printed in Great Britain

CARBON

AND NITROGEN
R. M.

NUTRITION
and C. B.

OF TRICHODERMA

DANIELSON*

DAVEY

Department

of Soil Science, North Carolina State University, Raleigh, North Carolina :U.S.A.
(Accepted 29 January 1973)

Summary-In buffered media Trichoderma grew best with r_-alanine, r_-aspartic acid, Lglutamic acid and casamino acids as sources of N. Growth on NH:-N was consistently superior to growth on NO;-N. Some isolates of T. koningii Oud. and T. hamatum (Bon.) Bain. were incapable of utilizing NO,. The best carbon sources were dextrose, fructose, mannose, galactose, xylose, ribose, trehalose and cellobiose. The ability to use melezitose, rathnose, sucrose and inulin as sources of carbon were related to taxonomic units. It is proposed that the ability to use these carbon sources as well as tests for extracellular oxidase, NO,- utilization and temperature maxima may be useful to delineate and identify species. All isolates tested readily decomposed pure cellulose but the time required to adapt to the cellulose medium varied considerably. The relative vigor of cellulose decomposition was not related to the loss in weight of pine needles or dogwood leaves induced by Trichoderma.

INTRODUCTION CLASSICAL taxonomy places the major emphasis of taxa differentiation upon reproductive isolation (Mayr, 1963) and morphological differences and similarities. However, when considering asexual organisms, the separation and definition of species becomes more difficult. These organisms often possess simple form and developmental patterns. Large groups of individuals of a genus assembled from diverse habitats may exhibit continuous variation in the size and shape of vegetative and reproductive structures. The task of defining whether subgroups of a genus are entitled to species status is difficult and the defining criteria are often chosen and utilized somewhat subjectively. If the species are real and not imagined, they should exhibit unique combinations of physiological and ecological traits as well as unique morphological features. Mayr (1963, p. 66) states that each species is a unique biological system and that no matter how similar two species may be morphologically, they will be found to differ in physiological preferences and tolerances. The revision of the genus Trichoderma by Rifai (1969) is an example of the difficulty in using asexual morphological criteria in defining species. Although Rifai could recognize different species of the perfect stage, Hypocrea, the morphological similarity of many of their asexual stages required the adoption of species aggregates rather than species for Trichoderma. The most important aspect in the nutrition of heterotrophic organisms are the carbon and nitrogen utilization patterns. The variety of substrates in nature provides an opportunity for the development of biochemical diversity among organisms. This is well illustrated by the diverse degradative ability of saprophytic microorganisms with variation existing between closely related, as well as distantly related, organisms. One method of separating species exhibiting small morphological differences, e.g. yeasts, is by nutritional tests. This paper describes the carbon and nitrogen assimilation patterns for a number of Trichoderma isolates and examines the consistency of the most promising * Present address: Department of Biology, University of Calgary, Calgary, Alberta. 505

506

R. M. DANIELSON

AND C. B. DAVEY

nutritional tests within a wide spectrum of isolates. The ability to degrade natural and ceilulose by different species is also tested.

substrates

MATERIALS AND METHODS

The basal medium used for the N-nutrition experiments was Yeast Carbon Base (Difco). Mineral N-sources and urea were added at a rate of 1.06g N/l and amino-N at 0.2 g N/l (with appropriate NH:-N controls). The media were buffered at pH 4.2-4.3 with O-1 N citrate buffer to give pH stability without inhibiting growth {Danielson, 1971). Filter-steriii~ed medium (25 ml) was dispensed into each 250-mI Erlenmeyer flask and three Aasks were included per treatment. Each flask was inoculated with one drop (about 0.05 ml) of a spore suspension ( IO6 spores/ml) washed from malt agar slants. Cultures were incubated for either 4 or 5 days on a reciprocating shaker. The pH of each culture was determined at harvest and then mycelium from each flask was collected on preweighed Whatman No. 31 filter paper and washed three times with distilled water (a total of 100 ml). The mycelium was dried for 24 h at 70C and weighed. The production of yellow pigments in both the C and N tests was noted as pigments or crystals may have antibiotic properties (Meyer, 1966). Carbon sources were added to nonbuffered Yeast Nitrogen Base (Difco) at a rate of 5 g/l to determine patterns of carbon utilization. All media, except soluble starch which was autoclaved, were filter-sterilized, Medium (25 ml) was dispensed into flasks (three replicates), inoculated with spore suspensions, and incubated under still conditions. The incubation period varied for each individual isolate and was determined by making daily harvests of dextrose cultures. When growth on the dextrose cultures approached maximum yields, all treatments for that particular isolate were harvested. Harvesting and dry weight determinations followed the same procedure as in the N tests. Additional tests using the same media were made to determine the consistency of utiiization of certain carbon sources. The media (3 ml/lOOx 13 mm test tube) were autoclaved, inoculated with spores taken from malt agar slants and incubated for 2 weeks. Nitrate utilization was determined in a similar manner using Yeast Carbon Base and 0.11 g N/I as KNO,. In addition, tests were made for extracellular polyphenol oxidase using gallic and tannic acids (Nobles, 1965). The ability of Trichodernza to degrade cellulose was tested using Whatman powdered cellulose (Chromedia CFI). Oven-dried cellulose (1.0 g) was placed in 50 ml of Yeast Nitrogen Base in 250-ml Erlenmeyer flasks. Following autoclaving, the flasks were inoculated with 0.15 ml of a spore suspension ( lo6 spores/ml). For comparison, 5 g of the F horizon from a loblolly pine stand was blended in 200 ml of sterile water. This suspension (1 ml/flask) was used as inoculum for an additional series of flasks. Noninoculated flasks served as controls. Flasks were incubated on a reciprocating shaker at 23-27C. After 8, 15, 30 and 40 days, three flasks of each isolate were harvested. The pH was determined and the mycelium plus the residual cellulose was collected on preweighed Whatman No. 42 filter papers. These were washed three times with a total of 50 ml of distilled water, dried at 70C for 24 h and weighed. The ability of Triclzodema to decompose natural substrates was tested by determining the weight loss of green and freshly fallen loblolly pine (Pintos raeda L.) needles and dogwood (Cormsf7orida L.) leaves following incubation with Trichoderwa. Green needles were collected from the tops of 30-yr-old loblolly pine trees and freshiy fallen brown needles and dogwood leaves were collected from a forest stand. The pine needles were air dried and cut into lengths of about 5 cm. Dogwood leaves were broken into small pieces (about 1 x 3 cm).

CARBON

AND

NITROGEN

NUTRITION

OF TRKHODERMA

507

Approximately 5 g of green needles, 3-4 g of brown needles or 1 g of dogwood leaves were placed in preweighed, 4-02 French square bottles. The material was dried to a constant weight at 70C and weighed. The needles and leaves were sterilized by autoclaving the dry material for 20 min. Sterile distilled water (15 ml) was added following autoclaving and the bottles were allowed to incubate for 3 days in order to ensure sterility and for the leaves and needles to become thoroughly moistened. In a second moisture regime, green needles were totally submerged in 75 ml water. One series of bottles was inoculated with 0.1 ml of a spore suspension (IO6 spores/ml). A second series was inoculated with 1 mf F horizon suspension (described above). Three replicates of each treatment were used. Three noninoculated bottles of each of the four treatments were harvested at the end of each incubation period to serve as controls. At the end of I-, 2-, and 3-month periods the cultures were dried to a constant weight at 70C and weight losses determined.
RESULTS

The reaction of the solutions remained in the range pH 4.0-4.5 until growth exceeded approximately 140 mg at which time the pH values rose sharply. Considering all isolates, growth was best with amino-N, NH: served as the next best source of N, followed by urea and NO; (Table 1). All isolates except 7. ~~~~~~i~~~r~~z Hammill grew well on urea. Nitrate was a decidedly inferior source of N for most of the isolates tested. One isolate of T. koningii Oud. did not grow at all on NO; while T. pol~~sporum (Link ex Pers.) Rifai, T. vivide Pers. ex S. F. Gray and one isolate of T. ~la~~ia~l~~l Rifai grew poorly. calanine,
Trichoderma

TABLE

1. GROWTH

OF

ON

NITRATE-, UREA-, AND NITROGEN

AMINO-NITROGEN

RELATIVE TO AMMONIUM-

Isolate

T. koningii (T-1) T. polysporum (T-9) T. harrianunt (T-33) T. ciride (T-67) T. ha~zff~u~z (T-14) T. kotringii (T-12) T. saturnisporum (T-74) T. hamatm (T-6) T. ~arzia~~f~?z (T-82) T. ~~e~d~ko~iFr~ii (T-49)
Average, all isolates

0 13 1st 24 48 48 71 80 81 89 47

81 87 90 75 73 102 12 101 77 100 71

120 200 1OOt 91 145 85 ND 202 ND 120 133

110 18.5 90 96 119 99 ND 224t ND tf6 130

125 238 138f 85 136 108 ND 41 ND 119 124

83 194 77t 68 Ill 73 ND 44 ND 1137 95

52 55 83t 16: 51$ 42 ND 26:: ND 14:: 42

29 14 64 23 39 21 ND 20 ND 291 30

37 14 53 9 50 11 ND 13 ND 5 24

24 17 8t 1 22 16 ND 16 ND 8 14

14 14 13 20 19 6 ND 9 ND I4 14

6 5 6 I 8 5 ND 4 ND 7 5

* Casamino acids assumed to be 10% N. f One or more solutions yellow. $ Mycelium with reddish tint. ND-Not determined.

508

R. M. DANIELSON

AND C. B. DAVEY

L-aspartic acid, L-glutamic acid and casamino acids generally served as excellent N-sources for Triclzoderma. The other amino acids were not utilized as effectively. L-cysteine, L-leucine and L-lysine were moderate to poor sources of N, while D-aspartic acid, L-histidine and particularly beta-alanine were poor N sources. Sodium nitrite (0.2 g N/l) was toxic in this acid system as evidenced by the lack of even a trace of growth. The production of yellow pigments was very erratic and bore no relationship to either substrate or the amount of growth.
(0) (0) (A) (A) T T T. T. virlde virlde koninqii koninqii NH4CI KNOJ - NH4CI - KNOs

Days FIG. 1.

of

incubation nitrogen

Growth of T. ciride and T. koningii on ammonium and nitrate (a) and changes in media pH (b).

(1.06 g N/l)

To determine if the poor yield of Trichoderma on NOT-N was due to differences in rates of growth, T. uiride and T. korzhgii were grown on NH:and NOT-N and yields determined at various time intervals (Fig. la). Both the growth rate and the total amount of growth was greatest with NH:-N. It is clear that if a single measurement of growth is used, comparative results are highly dependent on time due to differences in rate of growth. Further, if harvests are made after extended times of incubation, differences may not be apparent due to autolysis of the mycelium. The very rapid and extreme changes in solution pH is also readily apparent (Fig. lb). After 5 days in the media used here (buffered with citrate), alkalinity can limit growth. Clearly the citrate buffer is not an ideal buffer, as it is not nutritionally inert once growth exceeds a certain point. It contributes to, rather than prevents, changes in acidity in older cultures. In a medium lacking citrate and containing an NH:-N source, the pH decreased.

CARBON

AND

NITROGEN

NUTRITION

OF

TRICHODERMA

509

TABLE 2. GROWTH OF Trichodertna

STRAINS ON 3 I POTENTIAL CARBON SOURCES RELATIVE TO DEXTROSE

Carbon

source

(Per cent of yield with dextrose)

Monosaccharides D-Fructose D-Mannose D-Galactose

L-Sorbose L-Rhamnose L-Arabinose D-Arabinose

D-Xylose

o-Ribose Disaccharides Maltose Sucrose Lactose D-Cellobiose D-Melibiose D-Trehalose Trisaccharides Raffinose D-Melezitose
Alcohols Ethanol Glycerol i-Erythritol D-Sorbitol o-Mannitol Dulcitol i-Inositol Dextrose derivatives D-Glucosamine a-Methyl-d-glucoside Polysaccharides Dextrin Soluble starch Inulin Salicin Dextrose yield (mg) ND-Not determined.

49 99 109 13 12 24 12 116 63 60 100 34 96 69 86 20 0 13 42 66 19 80 12 5 17 6 80 50 0 31 59

32 97 98 16 14 24 18 134 39 11 0 3 102 31 75 19 0 11 101 56 75 90 5 1 23 4 72 79 0 22 39

63 97 84 28 21 34 21 94 88 15 0 26 98 46 83 7 0 54 11 79 77 105 17 7 18 9 61 35 0 48 52

78 102 92 16 8 57 9 97 42 48 105 67 95 63 6 40 39 33 73 54 49 93 76 10 6 0 70 41 0 48 60

96 75 101 26 21 46 26 94 73 11 82 45 80 46 63 44 0 30 81 99 100 76 40 7 17 4 73 47 0 24 50

105 ND 94 71 20 83 31 100 63 25 0 79 100 81 90 42 0 42 106 69 111 92 26 ND 21 4 103 59 0 23 41

ND ND 64 35 11 24 24 75 63 5 0 62 95 38 107 0 0 25 29 59 44 56 14 ND 15 0 34 44 0 23 42

ND ND 71 5 10 49 28 96 75 75 58 77 93 93 92 64 0 39 117 47 33 75 29 ND 26 0 137 85 0 39 33

106 82 93 49 8 39 48 75 117 9 0 3 120 51 63 17 0 0 24 85 77 66 10 ND 39 5 140 68 0 19 34

114 95 99 0 11 57 9 74 124 34 87 73 106 94 95 72 0 85 144 99 25 24 24 2 36 0 116 76 101 54 34

Carbon rurtrition Compounds which in general served as the sources of carbon for Trichoderrna equivalent to dextrose included fructose, mannose, galactose, xylose, ribose and cellobiose (Table 2). Trehalose also supported excellent growth with a single exception, T. harzianunz (T-82). This isolate also differed from all other Trichoderma isolates in that it readily sporulated in

510

R. M. DANIELSON

AND C. B. DAVEY

liquid-shake cultures. Isolate T-82 also grew far better in a medium containing a high concentration of NaCl than any other Trichoderttzaisolate tested {Danielson, 1971). This strain differed from the other isolate of T. harziattwtztested (T-33) in that it utilized sucrose and melezitose, while T-33 did not. One-half of the isolates tested were incapable of growing on sucrose but when it was utilized it was also a good carbon source. Compounds which supported moderate amounts of growth included L-arabinose, melibiose, i-erythritol, mannitol, dextrin and starch. Rhamnose, i-inositol and a-methyl-d-glucoside were utilized very poorly or not at all by all isolates. Only one of the ten isolates tested was capable of growing on melezitose or inulin. The remainder of the carbon sources tested supported poor to moderate growth depending on rhe particular isolate. Although the C-assimilation tests were standardized by harvesting at a relatively uniform physiological age (as judged by their growth on dextrose), comparisons between species is difficult. The most unambiguous comparisons are those in which the differences in growth are the most extreme, i.e. moderate to good growth versus no growth, as was the case with sucrose, rafflnose, melezitose and inulin. Tests with other isolates indicated that the variability of the biochemical responses was related to the amount of cultural and morphologica variation observed (Table 3). 2. r*iride is readily recognized by its rough-walled spores and its utilization pattern was very consistent. Typical isolates of T. /<ot~kgii, those which sporulated primarily at the margin of the plates with masses of blue-green spores, were also very consistent in reaction. However, the rarer, atypical strains produced a variety of reactions.
TABLE

3.

GROWTH

OF SPECIES AGGREGATES

AND INTRASPECIFIC GROUPS DIAGNOSTIC MEDIA

OF Trichoderma

ON POTENTIALLY

Reaction to: -Tannic acid Group

T. ciride T. Eongibrachiatum T. koningii (typical) 2. koningii (atypical) T. polysporum T. harrianunz T. hamatum slow growth (SI) 21/2I 717

Utilization of: -NO;

MeIezitose Raffinose Sucrose Gallic acid (Number of positive tests/number of isolates tested) o/21 o/7* O/S8 l/7 6110 29140 014 616 O/6 O/IS o/10*
O/21

017
O/58 O/7 l/l0 3140 O/4 414 616 O/15 Of14

58/58 217

O/IO 13140

Smooth apices (S) Clustered, fertile (Cl) Straight, exudate (St) Curly, exudate (Cu) * Medium yellow.

O/4 17117 616 IS/l5 12112

X/21 O/7 5X/58 517 lo/lo 39140 O/4 515 6i6 15115 14114

44144 7/7* llljlll 217 33/46 13/40 o/4 12112 6/6 15/15 20/0

2111 717 58/X+ 213 lo/r0 40140 4/4* 515 616 O/l5 14/14*

T. polysporunz varied in its utilization of sucrose and gallic acid as well as in its cultural properties; some strains produced abundant yellow, needle-shaped crystals, similar to those seen in some isolates of T. har~intwrz. Those isolates of T. po~.vsporuna producing yellow crystals were always gallic acid positive and produced yellow pigments in the NO; medium. However, isolates which did not produce crystals also occasionally reacted with gailic acid. Isolates of T. ~ot?gibra~/7~ff~~~z were readily recognized on the gallic acid medium Rifai which was not darkened but turned bright yellow.

CARBON

AND

NITROGEN

NUTRITION

OF I-RiCffODERMA

511

T. harzinnunz is an aggregate containing considerable cultural and physiological variation. Typical isolates have a maximum temperature of 36-38C (Danielson and Davey, 1973), do not utilize sucrose or react with tannic acid. Strains producing yellow crystals consistently grew well on sucrose and darkened the tannic acid medium. Another cultural strain could be distinguished by the definite zonate pattern of sporulation and a maximum temperature for growth of 33C. These isolates turned the tannic acid medium pink, grew poorly on raffinose and sucrose and released a yellow pigment into the NO; medium. Other isolates of T. /xzrzianunz produced a coconut-like odor similar to some T. vit-irkisolates, These were tannic acid- and sucrose-positi~~e and had a maximu~n temperature of 33C. The T. ~?~~~~~tz{~ (Bon.) Bain. aggregate was subdivided into five recognizable groups based principally on growth characteristics and conidiophore morphology. The first group (Sl) grew much slower on malt agar than typical isolates. The second group (S) had conidiophore apices lacking any exudate and thus appeared very fine and smooth. In the third group (Cl) the conidiophores were clustered in appearance; each conidiophore and its branches remained distinct from the others rather than forming a solid tuft. The tips were usually fertile. In the fourth group (St) the tips of the conidiophores were covered with exudate and nearly straight with occasional branching at acute angles. The conidiophore tips of the fifth group (Cu) were distinctly curly and covered with exudate. The large number of tips with their exudate often gave the colonies a whitish or grayish-green appearance. Each group showed consistent differences in their responses to the selected substrates. Of particular interest is the ability of two of the groups to grow on metezitose, which is unusual for other species of Triclrodernw, and the inability of one group to utilize NO;.
Weight loss qf cellulose and leatles

Ten isolahes of Triclrodernza, representing six species aggregates, degraded cellulose, but at different rates (Table 4). For exampie, after 2 weeks of incubation T. ~se~~o~o~i~g~~ was one of the weakest cellulose decomposers, but at the next sampling date, 2 weeks Later, it was the strongest. Further, those isolates which initially appeared to be the strongest cellulose decomposers, ultimately were among the weakest. At the end of 2 months the most active decomposers were T. pselmdokoningii, T, harzianunl and T. viride.

TABLE

4. DEGRADATION DETERMINED

OF

POWDERED

BY WEIGHT

LOSS IN

CELLULOSE BY Trichoderma AS LIQUID CULTURE

Days of incubation

8
Isolate

15 30 (Per cent weight loss) 0.6 2.1 4.5 8.3 6-7 6-6 7-I 9.4 7.6 2.8 0 2.4 8.2 7.7 11.3 9-2 9-6 10.6 14.1 14.9 16.1 0

60

T. hamatum (T-652) T. hamarum (T-690) T. polysporum (T-262) T. hamarum (T-454) T. koningii o-1 83) T. kon~~gjj(T-51 5) T. harzianum (T-33) T. viride (T-67) T. hauzianum(T-790) T. pseudokoningii (T-444)
Litter suspension

0 0

0.5 3.8 48 3.0 2.6 1.3 1.6

0.1 0

6.0 9.1 9.6 10.6 IO.8 11.4 14.4 15.6 16.8 20.6 6.5

512

R. M. DANIELSON

AND C. B. DAVEY

Cellulose medium inoculated with a mixed microflora from the forest floor resulted in a very long lag period and less weight loss than all but one of the cultures inoculated with
Tricltodertna.

The pattern of weight loss from pine needles and dogwood leaves was less variable than that found for pure cellulose. At least two isolates of each species were tested; representative data are reported in Table 5. Losses in weight occurred rapidly during the first month but thereafter the rate of weight loss slowed indicating that readily available materials had been utilized. Regardless of the substrate, the degradative ability of each species, relative to the other species tested, was similar. However, the substrates differed in their resistance to decomposition; fresh green needles being the least resistant. Surprisingly, totally submerged needles were readily decomposed although hyphal growth was limited to a thin pad floating on the water surface. Brown needles which had naturally fallen from trees were much more resistant to decomposition by Trichoderma. This would be expected as soluble substrates would be largely lost before leaf fall. Dogwood leaves were more rapidly decomposed by Trichoderma than freshly fallen loblolly needles. The species aggregates which produced the greatest weight losses of leaves were T. hamatum, T. koningii and T.karzianum.
TABLE

5.

WEIGHT

LOSS OF LOBLOLLYPINE NEEDLESAND DOGWOOD INCUBATION WITH Trichoderma

LEAVESDURING

Loblolly pine Months of incubation 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 3 Green submerged Brown Dogwood (Per cent weight loss) ND ND ND 2.2 5.2 7.5 ND ND 8.6 ND ND 7.4 3.9 7.0 8.2 ND ND 9.8 2.3 6.5 8.9 ND ND ND 8.4 ND ND ND ND ND ND ND ND 2.8 ND ND 3.4 1.7 2.9 2.8 2.6 3.4 3,9 2.4 3.4 3.9 ND ND ND 3.4 ND ND ND ND ND ND ND ND 3.8 ND ND 4.4 2.1 4.0 5.2 4.4 5.2 6.5 2.6 5.1 6.6 ND ND ND 5.3

Green

Isolate T. pseudokoningii (T-l 64)

3.0 4.4 5.9 5.0 7.1 8.5 5.7 7.1 9.6 7.3 8.6 9.7 6.4 9.9 10.8 10.0 12.3 13.9 7.7 12.3 14.7 13.8 16.6 19.7 11.2

T. pseudokoningii (T-444)

T. viride (T-67)

T. polysporum (T-262)

T. harziunum (T-33)

T. koningii (T-l 83)

T. hamatum (T-454)

Litter suspension Trichoderma average for 13 isolates ND-Not determined.

CARBON

AND

NITROGEN

NUTRITION

OF TRZCHODERMA

513

In contrast to the decomposition of cellulose, leaves inoculated with litter inoculum lost the most weight. The strongest decomposer of cellulose, T. pseudokoningii, was the weakest decomposer of leaves.
DISCUSSION

Growth of Trichoderma in buffered media was decidedly superior when it was supplied with NHf-N as compared to NO,-N. Some previous tests in nonbuffered media have indicated that NO; was a better source of N than NHf (Hacskaylo et al., 1954; Ward and Henry, 1961). The apparent superiority of NO; in those studies was almost certainly due to the production of inhibitory H-ion concentrations in cultures with NH:. Racle (1965) observed higher yields of Trichoderma mycelium with NO; than with NHf but determined that NH,+ was preferentially used when NH4N03 was the N-source. Aube and Gagnon (1969) tested three strains of Trichoderma on several sources of mineral N in nonbuffered media and found NHaN03 to be generally superior to either NO; or NH,+ alone. The length of the incubation period is critical when making single-harvest comparisons (Fig. 1). Ignoring either the time or acidity factor may make comparisons of N-utilization misleading or meaningless. Aube and Gagnon (1969) and Hacskaylo et al. (1954) found growth of Trichoderma to be better on asparagine than on mineral N. Certain amino acids were also found to be generally superior to NH,+ or NO; in this study although there was a good deal of variation among species. The best sources of amino-N for Trichoderma were alanine, aspartic acid, glutamic acid and casamino acids. This is in agreement with the general pattern of amino acid utilization by a wide variety of fungi (Cochrane, 1958). As would be expected for common soil saprophytes, Trichoderma species were capable of utilizing a wide variety of compounds as a sole source of carbon. A comparison of the results reported here with previous studies on the C-nutrition of Trichoderma reveal only minor differences. Aube and Gagnon (1969) reported poor utilization of xylose by one isolate. In contrast, xylose supported excellent growth on all isolates tested in this study and of those tested by Mandels and Reese (1957) and Ward and Henry (1961). Melezitose was utilized by only a small percentage of the isolates of Trichoderma from forest soils. A comparison with other fungi is difficult in that melezitose has been rarely tested, but Hesseltine et al. (1970) found utilization to be common in the Aspergillusjiavus-oryzae group. Utilization is probably common in other genera of soil fungi since melezitose is a major carbohydrate component of the canopy drip of some trees (Carlisle et al., 1966) and since utilization by forest soil bacteria is common (Goodfellow et al., 1968). Cochrane (1958) concluded that dulcitol, mannitol and erythritol are rarely utilized by fungi. This may not be true of soil saprophytes as these compounds were utilized by all the strains of Trichoderma tested in this study; in fact, the latter two alcohols supported good growth of most isolates. Cochrane (1958) also considered maltose a good carbon source, but most isolates of Trichoderma grew poorly on it. Previous studies using purified cellulose or carboxymethyl-cellulose have generally indicated that Trichoderma is one of the most active cellulose decomposers (e.g. Domsch and Gams, 1969). This is supported by this study where all the isolates tested were capable of extensive cellulose decomposition. However, there were large differences in the amount of time required for different species to adapt to the cellulose medium. Thus cellulolytic ability can be compared with respect to rate as well as total degradative ability after prolonged incubation. The important question, however, is whether this ability to degrade purified cellulose in culture is indicative of the behavior of the fungus in natural systems. Neither

514

R. M. DANIELSON

AND C. B. DAVEY

Hering (1967) nor Frankland (1969) could detect any change in the cellulose content of oak leaves or bracken petioles incubated with Trichoderma for 6 months. Similarly, Butcher (1968) could detect no weight loss of beech blocks incubated with Trichoderma. It appears that although Trichoderma rapidly decomposes purified cellulose, its ability to decompose natural cellulose in a noncompetitive situation is limited. This also suggests that purified cellulose in a medium with a rich N-source is not a useful model for predicting the ability of organisms to decompose cellulose in nature. This pure culture work does not take into account the competitive saprophytic ability of Trichoderma in a low-N environment. The production of yellow pigments and crystals by Trichoderma is common and not restricted to a single taxon. Masses of yellow crystals were produced by strains of T. harziar~um and T. polwporum. Rifai (1969) noted yellow or golden brown crystals in cultures of T. aureooiride Rifai and Slater et al. (1967) observed yellow crystals in agar medium with T. zliride Pers. ex Fries. Yellow or needle-shaped crystals possessing antibiotic properties have been extracted and precipitated from cultural fluids of the Triclloderma stage of H_Jpocrea schweinitzii (Fr.) Saccardo (Hashioka et al., 1961) and T. zliride Pers. ex Fries (Meyer, 1966). Although biochemical differentiation of species of yeast is an accepted practice, few studies have been done to test the usefulness of this technique with filamentous fungi. Hesseltine et al. (1970) attempted to separate species of theflutlus-oryzae group of the genus Aspergillus using C-assimilation patterns. They were unable to find any physiological criteria which would clearly separate species or strains. The principal problem did not appear to be excessive variation within morphological groups, but rather the sparsity of nonutilizable compounds. A similar situation exists for Trichoderma in that most of the compounds tested were utilized by all the isolates. However, sucrose, raffinose, melezitose, inulin and NO; supported good growth of some groups while others were unable to utilize them. In addition, a good deal of variation existed in the response of various groups of Trichoderma to tannic and gallic acid media. It appears that these nutritional tests combined with temperature response patterns (Danielson and Davey, 1973) would allow a physiological differentiation of some strains and species. Data such as these also indicate which groups are the most heterogeneous and thus most in need of further taxonomic analysis and simplification. Knowledge of the perfect stage combined with physiological data may provide a basis for separating morphologically similar species of imperfect fungi and a practical means of identifying them.
Ackno~~ledgernents-Paper No. 3717 of the Journal cultural Experiment Station, Raleigh, N.C. 27607. through contract A8fs-20147, supplement 23. Series of the North Carolina State University AgriThis work was supported by the US. Forest Service

REFERENCES
AUBE C. and

Effect of carbon and nitrogen on growth and sporulation of Trichoderma riride Pers. ex Fries. Can. J. Microbial. 5, 703-706. BUTCHER J. A. (1968) The ecology of fungi infecting untreated sapwood of Pinus radiata. Con. J. Bar. 46, 1577-l 589. CARLISLE A., BROWN A. H. F. and WHITE E. J. (1966) The organic matter and nutrient elements in the precipitation beneath a sessile oak (Qwrcus petraea) canopy. J. Ecol. 54, 87-98. COCHRANE F. W. (1958) Physiology ofFkgi. Wiley, New York. DANIELSON R. M. (1971) The Ecology and Physiology of Trichodcrma in Forest Soils. Ph.D. Thesis, North Carolina State University. DANIELSON R. M. and DAVEY C. B. (1973) Nonnutritional factors affecting growth of Trichoderma in culture. Soil Biol. Biochem. 5, 495-504. DOMSCH K. H. and CAMS W. (1969) Variability and potential of a soil fungus population to decompose pectin, xylan and carboxy-methylcellulose. Soil Biol. Biorhem. 1, 29-36.

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CARBON

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