Competition between Trichoderma species: Effects of temperature and litter type

Sod Bid. Biochm. Vol. 19. NO. I, pp. 89-93, 1987 Printed in Great Britain. Ail rights reserved

Copyright c

00380?17/%7 S3.00 + 0.00 1987 Pqamon JournalsLtd

COMPETITION BETWEEN ~~~C~O~~~~~ SEiECIES: EFFECTS OF TEMPERATURE AND LITTER TYPE PAUL WIDDEN

and DIANA HSU’

Concordia University, Biology Department. 1455 de Maisonneuve Canada H3G lM8

Blvd W., Montreal, Quebec,

(Accepred 12 July 1986)

Summary---The ability of five speciesof Trichoderma to competitively colonize maple and pine litter under a range of temperature conditions was evaluated and compared with published data on competition for spruce litter. It was shown that generally T. ~l~spo~m and T. viride competed most effectively under low temperature conditions (S-15°C). whereas r. hamatum. T. konigii, and an undescribed species. LP58. competed best at higher temperatures (2~25’C). Of the live species tested, T. koningii was an effective competitor over a wide range of temperatures, whereas LP 58 was not very effective under any of the experimental conditions. Litter type did not have much effect on the competitive ability of T. koningii or T. @ysporum. T. humatum tended to compete effectively over a wide range of temperatures for maple litter, and was a poorer competitor for pine litter than for mltple litter at 25X. ~ric~o~ermu sp. LPSS was a better cum~titor for maple litter than for pine litter. T. ciride wns shown to be a very poor competitor for maple litter, even at low temperatures. The comperitive abilities of T. knmutum, T. koningii, T. polysporum and T. hide, under the experimental conditions, appear to reflect the trends that emerge from field observations, whereas the data for LP58 conflict with field observations. These observations would suggest that the niche factors influencing the distribution of LP58 are somewhat different from those influencing the other species investigated.

in the deciduous forest soils and T. ooivin the spruce fore& &I. An intensive study of the spruce forest (Widden and Abitboi, 1980) showed that each species also had its preferred season when it was most abundant. Widden (1984) has shown that the effects of tem~rature on the ability to compete for spruce litter may play a role in this seasonal pattern within the spruce forest. This study was conducted to examine the effects of litter type on the ability of Trichodermn species to compete with one another under controlled temperature regimes. In this way it was intended to test the hypothesis that the observed preferences of Trichodermu species for particular forest soils may be explained by differences in their abilities to compete with one another for the types of litter available.

INTROIXCTlON

abundant

sporrrm was most abundant

Members of the genus Trichoderma arc known to be common inhabitants of soils and have a worldwide distribution (Domsch et ul.. 1980). with the possible exception of polar regions {Dowding and Widdcn, 1974; Widden and Parkinson. 1979). There has been much recent interest in Trichoderma species. because of their ability to produce cellulases (Mandcls, 1975; Domsch et al., 1980), and their possible role as agents of biological control (Baker and Cook, 1974; Harman et uf., 1980; Elad ef of., 19SI; Papavizas et al., 1982; Nelson and Hoitink, 1983). In spite of this interest, since the work of R. M. Danielson (unpublished Ph.D. thesis, Raleigh, N.C., 1971) and Danielson and Davey (1973 a, b, c) there has been relatively little work published on the ecology of Trichoderma species in natural soils, or on the factors that may affect the distribution of the various species in different soil systems. A comparison of the mycofloras of four forest’ plots in southern Quebec (Widdcn, 1979, i986a) demonstrated that the type of forest greatly influenced the distribution of individual microfungal species including species of Trichoderma. Trichoderma hum&urn was most abundant in two mixed deciduous (maple-hickory~ forest soils, T. riride and an undescribed species (LPSI) were most abundant in a pine and a spruce forest soil, T. koningii was most abundant in the pine forest soil, but was fairly ‘Present

address:

VF Peng Chau

Terrace.

iMATERiALS AND METHODS The cultures of Trichoderm~ used during this study were those used by Widden (1984), and had been isolated from forests in southern Quebec (Widden, 1979): T. hamntum (Bon.) Bain. (LP62; DAOM 16707), T. koningii Oud. (LP70; DAOM 167074), T. po/ysporum (Link ex Pers.) Rifai (LP60; DAOM 167065), T. riride (Bon.) Bain. Pers, ex S. F. Gray (LPSS; DAOM 167060) and an undescribed species, LP58 (DAOM 167063). Cultures have been deposited at the Biosystematics Research Institute, Ottawa. Cultures were maintained in the laboratory on 2% malt extract agar (MEA) and stored at 5%. Leaf litter from white pine (Pinus strobus L.) and sugar maple (Acer saccharum Marsh) was collected

6, Ho Lee St.

Hong Kong. 89

PALL WIDDEN

90

from the floor of the forests described in Widden (1979) in early May, freeze dried, and then stored at -2O’C in a desiccator until needed. Litter was sterilized by autoclaving at 12O’C for 15 min. Suspensions of conidia from sporuiating Trick0 derma cultures were prepared as described in Widden (1984). Competition experiments were performed on each possible pair of Trichoderma species on each litter in the following manner. Glass Petri dishes containing 50 g quartz sand were prepared and sterilized by autoclaving. Several sterile pine needles or strips of maple litter (2 x 30 mm) were than placed on the surface of the sand. The time taken for penetration of each litter type by each Trichoderma species in the absence of competition was tested at 5, 10, 15, 20 and 25°C. This was done by adding 8 ml of sterile water containing I x IO6 conidia of the test species to the dish containing the litter, incubating at the desired temperature, and periodically removing a piece of litter. The litter was surface sterilized with HgCI,. cut into 2 mm lengths. and fragments were plated on MEA and incubated at 20’C. When the Trichoderma could be regularly recovered in this manner it was judged to have successfully colonized the litter. Knowing the penetration times, competition experiments between each pair of Trichodermas were performed by inoculating I x IO” conidia into scparate dishes containing sand and 5 pieces of litter. For each test pair, 5 dishes wcrc prepared in which the conidia were separately added in the ratios of I :0, 0.8:0.2. 0.6:0.4, 0.4:0.6, 0.8:0.2, and 0: I (species A : species B) respectively. The dishes were held at the desired temperature for as long as it took the slowest of the pair alone to penetrate the litter, plus I extra week. After incubation, the litter was surface sterilized, cut into 2 mm segments and 5 segments from each piece of litter (25 in total) were plated separately on malt agar, incubated for I week at 20°C and the growth of Trichoderma species from the needle was recorded. Experimental procedures used are given in detail in Widden (1984). The only deviation from those procedures was in the surface sterilization of the maple litter. Because the maple litter is much thinner than pine or spruce litter, it proved necessary to reduce the concentration of HgC.1: from 0.1 to 0.016% and change the time of exposure from 2 to 3 min. The competition data were initially analysed by visual inspection of diagrams plotting the percentage of litter fragments colonized by each fungus against

and DIASAHsu the percentage of conidia of each species, Figs I-IO in Widden (1984). To obtain a more critical evaluation of the results, and to save space in presentation of the data in this study, the data were analysed using the x2 test (Sokal and Rohlf, 1981). The x2 test was applied separately for each test involving mixtures of conidia within an experiment, setting the expected frequency for recovering each species from the needles proportional to the percentage of conidia in the mixture. Where more than one species was recovered from a litter fragment, each species was scored as 0.5. The sums of the deviations from expected, before squaring, were also calculated in order to gain an index of the magnitude of the deviation from expected in favour of one or the other species, across the four tests that made up each experiment. Because each test used 25 litter pieces, the maximum deviation from the expected, across each experiment was +50 in favor of either species. RESULTS

The penetration times for each species of Trichoderma on each litter type are given in Table I. In general, they show that penetration time decreased as temperature increased. They also indicate that. at SC pint litter took longer to penetrate than did maple litter, with the exception that T. uiride penetrated pine litter more rapidly than maple. It was also notable that on maple litter, i? airide took longer to penetrate at 25 C than at 20 C. The results of the competition experiments are presented in Figs l-10. which give the total deviation from the expected frequency in favor of one or the other species in the species pair. It is clear from these data that both the type of substrate and temperature had a large influence on the outcome of the competition. In only one case (T. koningii and T. airide) was the outcome unaffected by these factors, with T. koningii outcompeting T. uiride at all temperatures and on both substrates (Fig. 7). In some cases (e.g. T. polysporum vs T. hamatum, T. koningii, or LP58). on both substrates, the effect of temperature on the outcome of competition is the same, thus, in all of these examples, T. polysporum competes successfully at low temperatures (Figs 2, 5 and 8). In the case of T. hamnfum or T. riride, the temperature response differs with regard to substrate, thus on maple litter T. humufurn is a better competitor against T. chide at any temperature, whereas on pine litter, it is only a better competitor at high temperatures (Fig. 4);

Table I. Appronimak penc~ntmn hmes (days) l’or Trrrhod~mro specieson pint and maDIe liltrr Lllllx type

SC

IO c

IS C

Pine Milpk

127 30

25 JI

IO 29

IX

Pine khpk

57 30

27 27

19 18

8 18

8 7

Pine Maple

96 26

25 27

17 25

I4 22

14 a

7-. ciride

Pine Maple

35 46

2s 41

IO 26

7 II

7 33

LPSX

Pine Maple

I27 50

30 24

25 29

25 21

27 I5

Species

7. Irumotum T. kuninyii

T.

pol~.rporum

20 c 7

25 c 7 II

Com~tition

33 2340

between Trichodemta species

34 24

05

91

42 4C t4 44 23

2 32 31 44 13

‘43 tC 3C 34 24

‘43 42 3C 32 33

I-

Go

07

50 too w

20=25*

&

+

08 r.‘,

+

LP 58

be 44

23 24 43 34

010 LP+&

C 33 20 CC 24

Figs I-IO. ERects of temperature and litter type on the competitive colonization by Trichoderma species. Bars represent the total deviations from expected in favor of one or the other species (maximum of 50). assuming that in the absence of competition fungi will be recovered from the litter in the same proportions as there were conidia present. Numbers below the bars represent number of significant y2 tests, C indicates contradictory results. Solid bars represent competition for pine litter, open bars for maple litter. T. ha = 7’. hamatum: T. ko = T. koningii; T. po = T. polysporum; T. ve = T. viride; LP58-an undescribed species.

Table 2. Summarv of nsul~s from comoerition cxtxrimcnts. Dalr a~ number of “wins” (4aximum 4) Spzcia

Substrate

5°C

10°C

IS-C

20%

2x

T. hamttum

Pine Maple

I’ 2

2w 2

I (I)6 2(2f

4 4

311) 4

1. koningii

Pine Maple

2 3

3(t) 3(l)

2

3 3

3 2

‘I. po!vspo?u~

Pine Maple

3 4

2 2(2)

Pine Maple

2(2) O(1)

3 0

&I, I(1)

i(3)

T. ciridc

O(1)

O(2)

&I,

LP58

Pine M&C

WC 0111

0 0

30) 2 (21

0 (2)

1w 2(l)

I(9 If3 1ut IO)

l(l)

‘No data for compelition for pine litter at J”C between TI hamatum and LPSB. bNumbcrs in brackets represent tics or contradictory results. Wo data for competition for pine litter at J”C between 7’. uirideand LP58.

PAUL WIDDEN

92

against I: koningii the reverse is true, as T. hamatum competes well at all temperatures on pine litter. but only at higher temperatures on maple litter (Fig. 1). The general trends in the data are summarized in Table 2, which gives the number of “wins” for each species at each temperature and on each substrate. From these data it is clear that T. hamatum competes best at 20 and 25’C. T. polysporum at temperatures of 5 and IO’C and T. koningii is a fairly good competitor over a broad range of temperatures. LP58 was a good competitor at intermediate temperatures, particuiarly on pine. and T. uiride is a fairly good competitor for pine litter at 5 and IO”C, but is not a good competitor for maple litter at any temperature.

DISCUSSION

The results of these experiments have confirmed the general picture of the effects of temperature on competition between these species of Trichoderma that emerged from a study of Norway spruce litter (Widden, 1984), that is that T. polysporum and T. ciride tend to be better competitors at lower temperatures than high temperatures, whereas the reverse is true for T. hamatum and LP58. They also confirm the fact that T. koningii has good competitive abilities over a wide range of temperatures, and that LP58 is not generally a good competitor. The differences in bchaviour of the Trichodcrmas in relation to litter type that are revealed by the data are of considerable interest. For most species, the general pattern is similar on all litter types. but differs in detail. For T. ciride. however, there is a great effect of litter type on compctitivc ability; on pine litter it is a fairly good competitor over a wide range of temperatures, on maple litter it appears not to bc a good competitor at any of the tcmpcraturcs tcstcd whereas for spruce litter (Widdcn, 1984) it only competed well at low tcmpcraturcs. These data suggest that T. ciride should not bc the dominant Trichodernra in deciduous forests, but may be cxpetted to be more abundant in conifer forests, particularly during the cooler months. Field studies have shown that T. ciride is more abundant in conifer forests than deciduous forests in southern Quebec (Widden, 1979) but these differences were not statistically significant. Multivariate analysis of these data also revealed a tendency for T. ciride to bc more abundant in the conifer forest soils and to be associated with spring and fall conditions (Widden, 1986a, b). The behaviour of T. hamalum is also of some interest. Although the data show that T. hamalum competes best for pine and maple litter at warmer temperatures. there is some indication that T. hamatum is a better competitor at low temperatures for maple litter than it was on pine or spruce (Widden. 1984). and that at 25’C it was not as good a competitor for pine litter as T. koningii (Table 2). The combination of a moderate competitive ability at lower temperatures with the ability to outcompete all other Trichodermas tested at temperatures of 20 and 25’C is in line with field observations that T. humalum is the dominant Trichoderma in mixed hardwood forest soils (Widden. 1979). particularly in

and DIANA Hsu warmer periods (Widden. 1986b), and its poor competitive ability for pine litter at 25XZ may explain why it was less abundant in a pine forest soil than spruce or deciduous forests (Widden, 1979). The data for T. polysporum show that on maple and pine litter there was a competitive advantage only at low temperatures (5 and IO’C), whereas for spruce litter there is also good competitive ability at 15’C (Widden. 1984). This is again in accordance with field data that suggest T. pofysporum is most abundant in spruce forests (Sbderstrbm, 1975, S6derstriim and BUth, 1978; Widden, 1979) and in cooler soils (Danielson, 1971; SiiderstrGm and BGth, 1978; Widden and Abitbol, 1980). The data on T. koningii and LP58 are hard to relate to field studies. The ability of T. koningii to compete fairly successfully for all substrates tested. over a wide range of temperatures would suggest that this species should be an abundant Trichoderma in many forest soils, over a wide range of conditions. This agrees with Danielson’s (1971) conclusion that T. koningii is widely distributed, but in most studies where quantitative data for forest soil microfungi have been given, T. koningii is usually not a dominant species, though it may be abundant (SBderstriim and BGth, 1978; Widden, 1979; Widden and Abitbol, 1980). The behaviour of LP58 does not seem to bear much relation to the field observations that have been made. It was found to be abundant in pine, spruce and deciduous forest soils (Widden, 1979) and was a dominant in conifer soils, particularly spruce soils during summer (Widden and Abitbol, 1980). Although the laboratory data show that LP 58 grows very rapidly at high temperatures (Widden. 1984) and that it is a better competitor at higher tcmperaturcs (Table 2), it cannot outcompete T. hamatum or T. koningii at high tcmpcratures for spruce or pint litters. On the other hand, LP 58 was a good compctitor for maple litter at high tcmpcraturcs (Table 2). yet field data suggest that it is more abundant in conifer soils (Widdcn, 1979). The fact that LP58 has consistently given results that do not fit with the pattern shown by other Trichodermu species in this study suggests that it differs ecologically from the other species. Its comparatively poor competitive ability, coupled with very rapid growth rates (Widden, 1984) and fast, heavy sporulation (personal observations) suggest that it may be a more opportunistic or ruderal (Grime. 1977) spccics than the other Trichodermu species. The data presented here emphasize the importance of tempcraturc conditions and substrate type on the competitive abilities of Trichodermu species, and in a broader context. the importance of the environment in dctcrmining the outcome of competition. Clearly, many other factors can and will affect the ability of Trichodermas to compete for substrdtcs, for instance moisture, nutrient availability, pH, and the amount of prior decomposition of the substrate. Competition from sympatric species of Trichoderma will, in nature. bc only one aspect of the competitive pressures on any specks, as many other fungi, plus bacteria and the soil fauna will also compete for available substrates. Nevertheless, the experiments described here do illustrate how a simple model system can be used to explore the possible role of competitive inter-

Competition between ~richoderma species

actions in determining the distribution of species of fungi. A number of researchers are now experimenting with the use of Trichoderma species as biological control agents (Baker and Cook, 1974; Harman et uf.. 1980; Elad et uf., 1981; Marois et al., 1981; Papavizas et al., 1982; Henis et al., 1983; Nelson and Hoitink, 1983). If such attempts are to be

made in a rational manner, a thorough understanding of the various factors affecting the distribution and abundance of Trichoderma species, including the importance of such interactions as have been described in this paper, is essential. Ackno,rledgmrenu-We thank Michelle Groleau and Cornelia Karkosa for their technical assistance. This project was financed by a grant to the senior author from the Natural Sciences and Engineering Research Council of Canada. REFERENCES

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