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The effects of dictyostelids on the formation and maturation of myxomycete plasmodia

     Department of Biology, University of Kentucky, Lexington, Kentucky 40506

John C. Landolt

     Department of Biology, Shepherd College, Shepherdstown, West Virginia 25443

Steven L. Stephenson

     Department of Biology, Fairmont State College, Fairmont, West Virginia 26554

	ABSTRACT

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Dictyostelids (cellular slime molds) and myxomycetes (plasmodial slime molds) are two groups of mycetozoans usually present and often abundant in the soil and litter microhabitats of terrestrial ecosystems. Because they utilize the same food resource and occur together in a spatially limited and clearly defined microhabitat, the potential for ecological interactions would seem to exist. However, relatively few previous studies have considered this aspect of mycetozoan ecology. In the present study twenty-eight isolates (8 species) of dictyostelids were co-cultured in all possible pair-wise combinations with fourteen isolates (7 species) of myxomycetes to determine if there were any effects on the production of fruiting bodies. Dictyostelids showed little or no delay in culmination and only random and inconsistent reductions in sorocarp abundance when co-cultured with myxomycetes. In contrast, myxomycetes displayed a number of specific effects. The heterothallic isolates exhibited delays in plasmodial formation and/or maturation, with some pairings showing little to no effect, while others displayed nearly complete inhibition of plasmodial formation or maturation. Apomictic isolates, in general, were much less affected, with only a few combinations displaying significant delays in both formation and maturation of plasmodia.

Key words: apomixsis, bacterivore, competition, heterothallism, mycetozoan

	INTRODUCTION

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Dictyostelids (cellular slime molds) and myxomycetes (plasmodial slime molds), along with soil amoebae, ciliates, and nematodes, are the major bacterivores in the soil and litter microhabitats of terrestrial ecosystems. Although the sporocarps of myxomycetes are abundant on coarse woody debris (Stephenson 1988), their myxamoebae make up a relatively small component of the bacterivore community in many forest soils, where dictyostelids are often abundant (Feest and Madelin 1988a). On the other hand, this situation is reversed in many non-forest soils, where myxomycetes (mostly species of Didymium) make up a substantial component of the bacterivore community (Feest and Madelin 1988b). In a study of the vertical distribution of the disctyostelids and myxomycetes in the soil/litter microhabitat of a deciduous forest in southwestern Virginia (Stephenson and Landolt 1996), densities of both groups were found to be positively correlated with numbers of bacteria at every level, but a coincident response to high (or low) bacterial numbers never existed for both groups at the same level. This type of inverse correlation of myxomycete and dictyostelid abundance appears to suggest that competition and/or resource partitioning could be occurring between these two groups of mycetozoans. Because they use the same food resource and occur together in a spatially limited and clearly defined microhabitat, the potential for ecological interactions would seem to exist. However, relatively few previous studies have considered this aspect of mycetozoan ecology, and virtually nothing is known about interactions between two groups or among mycetozoans and the various bacterivores that occur in the same microhabitats. Clearly, this is a system where basic field and laboratory investigations are needed.

While dictyostelid amoebae form sorocarps by means of aggregation and culmination, a myxomycete sporocarp is produced from a multinucleate plasmodium that develops from myxamoebae, either by means of heterothallic sexual fusion or by apogamic conversion (Collins 1979). Since mycetozoan abundance is determined (Feest and Madelin 1985) from the occurrence of dictyostelid sorocarps (a single amoeba in a soil dilution can produce an aggregation) and/or myxomycete plasmodia (apogamic development in a clonal population derived from a single amoeba, or sexual fusion between heterothallic amoeboid clones of different mating types) obtained from a single small sample of soil/litter, these estimates could be influenced by interactions between the two groups that affect amoeboid growth or conversion. The objective of this report is to investigate the effects of dictyostelid co-culture on the formation and maturation of myxomycete plasmodia.

	MATERIALS AND METHODS

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Twenty-eight isolates (8 species) of dictyostelids, 5 heterothallic myxomycete isolates (3 species), and 9 apogamic isolates (6 species) were re-isolated and cultured on three different bacterial food sources (Table I). These bacterial strains were selected due to their differential ability to support growth for several different isolates of dictyostelids and myxomycetes, with strain A generally supporting the most rapid and strain B the least rapid growth. The cultures were grown in 100 x 15 mm Petri dishes containing one of six agar variations: CM/2 agar (8 g Difco corn meal agar and 8 g plain agar per L of distilled water), CM/2⁺ (8 g corn meal agar and 12 g plain agar), CM/2^- (8 g corn meal agar and 5 g plain agar), CM/4 (4 g corn meal agar and 12 g plain agar), CM/8 (2 g corn meal agar and 14 g plain agar), and CM0 (16 g of plain agar). All co-culture tests were conducted in a standard manner for each pairing of a myxomycete and a dictyostelid. In each Petri dish there was a myxomycete control, a dictyostelid control, and a mixed culture test. Heterothallic myxomycete isolates were crossed by mixing a loopful of amoebae (from actively growing cultures) of each mating type clone in each of two 2.5-cm diameter spots. One spot served as the myxomycete control and two loopfuls of dictyostelid amoebae were added to the other spot to produce the co-culture test. A separate dictyostelid control was set up as a third spot on the plate. Apomictic tests were similar except that crosses of the myxomycetes were not needed. Each pairing was replicated three times and the plates observed daily with a 60x dissecting microscope for sorocarp and/or plasmodial formation and maturation. Plasmodial maturation was defined as the plasmodium expanding beyond the original spot inoculum.

	RESULTS

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Standard conditions – Co-culture of the 28 dictyostelid isolates with the 14 myxomycete isolates in all possible pair-wise combinations on CM/2 agar with bacterial strain A as the food source (Tables II and III).

Apomictic myxomycetes. The apogamic myxomycetes generally displayed little (one day or less) or no delay in plasmodial formation when co-cultured with dictyostelids (Table II). While a few dictyostelid isolates (Dl-2, Pv-5) had a relatively stronger general effect than other isolates, and some myxomycete isolates (Di-3, Pg-1, Pc-1) were affected more easily than others, many of the delays appeared to be sporadic and relatively minor in occurrence However, some combinations (Di-3 paired with any Da isolate, Pg-1 paired with any Dd isolate) produced consistent delays of a day or longer. Plasmodia maturation delay results matched and were consistent with the plasmodia formation results (not shown).

Heterothallic myxomycetes. The heterothallic isolates were in general more strongly affected than the apogamic isolates when co-cultured with dictyostelids (Table III). While the Didymium iridis Di-4 and Di-5 isolate crosses showed little or no plasmodial formation delay, except for the Dir-5 isolate paired with any Da isolate (which consistently displayed delays of a day or more), the Didymium iridis Di-6, Stemonitis flavogenita Sf-1, and especially the Didymium ovoideum Do-1 isolate crosses were often delayed or completely inhibited from forming plasmodia. Plasmodial maturation, in these same tests, displayed similar patterns with even more pronounced effects, with many of the plasmodia never reaching maturity (not shown).

Nutritional variations – Myxomycete plasmodial formation and dictyostelid sorocarp formation were compared in separate control and co-culture trials on different levels of nutrient agar. The standard medium (CM/2) for myxomycete culture is also suitable (but not standard) for dictyostelid culture. Therefore, trials were conducted using a series of different strength corn meal agar media: CM/2 ( strength CM), CM/4, CM/8 and CM0 (water agar). Eight dictyostelid isolates (Al-1, Da-2, Dd-1, Dg-1, Dl-1, Dmi-1, Dm-3, Pv-1) and four myxomycete isolates (Di-3, Di-4, Do-1, Dms-1) were used in this experiment.

Dictyostelids. No change in sorocarp formation was detected in nutrient level co-cultures compared to the standard conditions, at any nutrient level, except for a small general increase in sporangial density that was observed with increased nutrient levels for all dictyostelids except Acytostelium leptosomum, which grew best at the lower nutrient levels.

Myxomycetes. Minor (one day or less) increased delays in plasmodial formation were detected with higher nutrient levels in three pairings (Di-3 with Dg-1, Do-1 with Dg-1, and Dms-1 with Dm-3). However, there was no evidence for a general trend, and these minor effects are quite likely due to specific interactions or random variations. Similar patterns were found when plasmodial maturation was followed.

Agar hardness variations – The isolates used in the nutrient variation experiment were also used in the agar hardness experiment. The CM agar media was modified to contain 2.3% (CM/2⁺), 1.5% (CM/2) and 0.9% (CM/2^-) agar. As the percentage of agar goes up, the availability of water decreases. To keep the plates within their water ranges, the 0.9% plates had autoclaved distilled water added to them every day while the 1.5% plates had water added every other day.

Dictyostelids. No changes in sorocarp formation were detected in agar hardness co-cultures compared to the standard conditions.

Myxomycetes. Minor increased delays in plasmodial formation were noted with decreasing hardness in four pairings (Di-3 with Dm-3, Dms-1 with Dm-3, Dms-1 with Da-1, and Di-3 with Dl-1). Once again, there was no evidence for a general trend, and these minor effects were possibly due to random variations. Similar results were found with plasmodial maturation studies.

Bacterial variations – The same isolates used in the nutrient and hardness variation experiments were also used in a bacterial food source experiment. Three different bacterial strains (A, B, and P) were selected in preliminary tests because they supported different growth rates for a number of dictyostelids and myxomycetes. In general, as already noted, strain A supported the most rapid and strain B the least rapid growth.

Dictyostelids. Sorocarp formation, in general, was slightly accelerated in the B bacterial co-culture tests as compared to standard conditions, while there was no effect in the A and P bacterial cultures. These B cultures also seemed to produce fewer sorocarps than the A and P cultures.

Myxomycetes. No trends in increased plasmodia formation delay were seen on the different bacterial food source cultures. However, occasional non-formation of plasmodia occurred in the P and especially in the B bacterial tests for the Di-3 with Dmi-1 pairings. These results were even more apparent when plasmodial maturation was considered. The plasmodia, especially in the B strain cultures, often did not reach maturity in either the control or test situations for the myxomycete (Di-3) in these pairings.

	DISCUSSION

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Dictyostelids, with the exception of Acytostelium leptosomum, are not affected, in terms of the speed of formation and numbers of sorocarps produced, when co-cultured with myxomycetes. Apparently their rapid growth exempts them from possible detrimental effects that could be caused by the slower growing myxomycetes. Acytostelium leptosomum, whose growth was decreased in all co-cultures except for the very low nutrient situation, is different from the rest of the dictyostelids and is apparently adapted to slower growth on low nutrient substrates as a method of avoiding competition.

Members of the genus Acytostelium are the smallest of all dictyostelids and produce extremely delicate sorocarps. They are found less frequently in nature than members of the two other genera (Dictyostelium and Polysphondylium) recognized for the group. Although the localities from which the various species of Acytostelium have been isolated encompass a considerable range of ecological diversity, only two (one of which is A. leptosomum) of the eight species described thus far have been recorded outside the tropics, and the genus is rarely recorded from excessively dry or moist situations (Cavender and Vardell 2000). All of this would suggest that Acytostelium is less tolerant of environmental extremes and possible more susceptible to perturbations than most other dictyostelids. The data obtained in the present study would be consistent with such a hypothesis.

While there are specific interactions between particular myxomycetes and dictyostelids (Dms-1 with all Pv isolates, Table II), most of the observed effects in each pairing are apparently a property of the myxomycete component. Individual strains and species of dictyostelids all produce comparable average delays (0.5 to 0.8 days in Table II) in plasmodial formation. On the other hand, the average delay for the myxomycete strains ranged from 0.1 to 1.7 d. Apparently, there is a relatively general effect of dictyostelids on plasmodial formation and maturation, with different myxomycete isolates having different susceptibilities to this general effect. This pattern remained stable in a series of experiments that attempted to mimic the possible major environmental variables in the soil/litter habitat: variations in nutrient level, water availability, and the bacterial strain present. Although some minor differences were found, the myxomycete strain remained the major determinant of plasmodial formation and maturation variations in co-culture with dictyostelids.

Plasmodial formation requires the growth of the amoebae population and their conversion to competent cells after a certain density is reached (Youngman et al 1977). In apogamic isolates, the competent cells convert directly into plasmodia, while competent heterothallic cells undergo syngamy to produce a zygote that then develops into the large multinucleate plasmodium. Plasmodial formation could be affected, in both cases, by a delay or failure to reach competent cell densities (direct competition for food or a production of a toxin by the dicytostelids) or, in the heterothallic isolates, by blockage of syngamy (production of an inhibitor by the dictyostelids). The inhibition of plasmodial maturation, however, would appear to be due to a toxin, since there is no lack of nutrients for the plasmodia in the plates (plasmodia can ingest both their own and the dictyostelid amoebae).

Since the heterothallic myxomycete isolates generally display greater delays in plasmodial formation and development than the apogamic isolates, it is possible that the critical syngamy processes are the major target affected by the dicytostelids. This is supported by the much greater effects shown for the Stemonitis flavogenita (Sf-1) heterothallic line than in the apomictic line of this isolate (Tables II and III). However, rapidity of plasmodial formation is another factor that may help explain some of the differences. The average plasmodial formation times for the controls (not shown in the tables) ranged from 2.0 to 6.6 d for the various myxomycete isolates, and there is a inverse correlation with the degree of delay and the speed of formation. Pg-1 (apomictic) and Do-1 (heterothallic) have the longest developmental times for their classes and also are the most delayed in co-cultures with dicytostelids. Quick plasmodial formation, regardless of crossing or apomixis, appears to provide the greatest protection against the effects of dictyostelids in co-culture.

Hagiwara and Someya (1992) have reported that dictyostelids often exhibit a killer activity between the various species and strains. Therefore, this factor may contribute to their apparent toxicity to myxomycetes, which could explain the relatively low levels of myxomycetes isolated from forest soils (inhibition by the abundant dictyostelids). However, the abundance of myxomycetes on coarse woody debris and in certain non-forest soils indicates that this negative effect of dictyostelids on myxomycetes does not extend to all of the microhabitats in which the two groups of mycetozoans occur.

	ACKNOWLEDGMENTS

 
The majority of the dictyostelid isolates used in this study were obtained during the course of research funded in part by the USDA Forest Service under cooperative agreements 42-564 and 42-581 to SLS and JCL.

	FOOTNOTES

 
¹ Corresponding author, jdclar0{at}uky.edu

Accepted for publication April 24, 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Cavender JC, Vadell EM., 2000 The genus Acytostelium. Mycologia 92:992-1008

Collins OR., 1979 Myxomycete biosystematics: some recent developments and future research opportunities. Bot Rev 45:145-201

Feest A, Madelin MF., 1985 A method for the enumeration of Myxomycetes in soils and its application to a wide range of soils. FEMS Microbiol Ecol 31:103-109

———, ———. 1988a Seasonal population changes of Myxomycetes and associated organisms in four woodland soils. FEMS Microbiol Ecol 53:133-140

———, ———. 1988b Seasonal population changes of Myxomycetes and associated organisms in five non-woodland soils, and correlations between their numbers and soil characteristics. FEMS Microbiol Ecol 53:141-152

Hagiwara H, Someya A., 1992 Killer activity observed in dictyostelid cellular slime molds. Bull Nat Sci Tokyo Series B 18:17-22

Stephenson SL., 1988 Distribution and ecology of Myxomycetes in temperate forest. I. Patterns of occurrence in the upland forests of southwestern Virginia. Can J Bot 66:2187-2207

———, Landolt JC., 1996 The vertical distribution of dictyostelids and myxomycetes in the soil/litter microhabitat. Nova Hedwigia 62:105-117

Youngman P, Adler P, Shinnick T, Holt C., 1977 An extracellular inducer of asexual plasmodial formation in Physarum polycephalum. Proc Nat Acad Sci 74:1120-1124[Abstract/Free Full Text]

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