A METHOD FOR THE ENUMERATION OF MYXOMYCETES IN SOILS AND ITS

FEMS Microbiology Ecology 31 (1985) 103-109 Published by Elsevier

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FEC 0014

A method for the enumeration of myxomycetes in soils and its application to a wide range of soils (Myxogastrids; slime moulds; most-probable-number method)

A. Feest and M.F. Madelin Department of Botany, The Unioersityof Bristol, BristolBS8 1UG, U.K. Received 8 February 1985 Accepted 22 February 1985

1. SUMMARY A standardised most-probable-number technique for estimating the number of myxomycete plasmodium-forming units (PFUs) in soils has been developed and tested experimentally. Application of the method to a local soil showed that the uppermost layers contained the most PFUs. Its application to 44 surface softs from various parts of the world detected myxomycetes in 36, of which 4 were desert soils. In general, highest numbers of PFUs (up to 9000. g-1 soil) were recorded in grassland and agricultural softs. The nature of the PFUs has not been definitely resolved but they are probably myxamoebae, myxoflageUates and microcysts.

2. INTRODUCTION The literature relating to the geographical distribution of myxomycetes (myxogastrids or acellular slime moulds) suggests that they are nearly ubiquitous throughout the vegetated land masses of the world. Little is known about their actual ecological role, because the literature on the ecology of these organisms is almost wholly concerned with records of the natural occurrence of their

reproductive structures, rather than with their trophic phases [1,2]. There is likewise little information on the natural occurrence of myxomycetes in soils, though there are reports that indicate that soft is a habitat for some [3-7]. In the course of investigating the ecological role of myxomycetes in soft, we had need of a technique for their selective isolation and quantification. We here describe such a technique, based on the most-probable-number method, and report the numbers of myxomycetes detected in a variety of soils from diverse locations.

3. METHODS Half-strength corn meal agar (CMA/2) was prepared by dissolving 8.5 g of Oxoid Corn Meal Agar (Oxoid CM 103) and 7.5 g of Oxoid Purified Agar (Oxoid L 28) in 1 1 distilled water. Saccharomyces cerevisiae (Culture No. Y37 from the culture collection of the University of Bristol, Department of Botany), used as food for myxomycetes, was grown in Sabouraud's dextrose broth (Oxoid CM 147) for 4 days at 30°C in shake culture, harvested by centrifugation, washed 3 times by repeated suspension in sterile distilled water and centrifugation, and finally prepared as a

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standard suspension containing 1% packed cell volume. To prepare C M A / 2 plus yeast, 0.5 cm 3 standard yeast suspension was added to each 9-cm diameter petri dish containing approximately 20 cm 3 of solidified sterile C M A / 2 . Media were sterilised in an autoclave at 121°C for 20 min.

4. RESULTS A N D DISCUSSION

4.1. Standard method for enumerating myxomycetes in a soil sample In the light of experimentation and extensive practical experience gained over numerous trials, a standard procedure based on the most-probablenumber technique [8-12] was developed for enumerating myxomycetes in a given soil sample. The unit which we have adopted for the expression of myxomycete abundance in soil is the 'plasmodium-forming unit' (PFU) analogous to the colony-forming unit of filamentous fungi. The nature of PFUs and the interpretation of their abundance are discussed below. It was necessary that the medium adopted for the most-probablenumber technique should promote the appearance of myxomycete plasmodia in mixed cultures, since it was by the production of plasmodia that myxomycetes in the soil inoculum were recognised unequivocally. Since plasmodia appear able to live wholly phagotrophically [2], weak culture media with an overlay of washed cells of S. cerevisiae were used to discourage osmotrophic competitors. S. cerevisiae is suitable for the nourishment of plasmodia of numerous myxomycetes [13]. To satisfy the needs of the preceding phagotrophic unicellular phases, reliance was placed on bacteria derived from those inevitably introduced in the soil inoculum. Weak underlying agar media tested included 1.5% plain agar, corn meal agar, halfstrength corn meal agar and apple bark extract agar, singly and in combination, but none of these gave greater recoveries from soil than C M A / 2 with yeast overlay. The standard procedure was as follows. The collected soil sample was thoroughly mixed in a sterile plastic bag by kneading and shaking, and a sub-sample of 50 cm 3 was removed. The packing of the measured sub-sample was made to ap-

proximate to the density of the soil in its natural state. It was weighed and added to 450 cm 3 sterile 0.1% Brij 35 solution (a wetting agent; polyoxyethylene lauryl ether; B.D.H. Ltd., Poole, U.K.) in a conical flask and agitated thoroughly for 20 min on a wrist-action shaker to yield a 10 -1 dilution. These initial operations were scaled down for smaller soil samples. A further small sub-sample of soil was oven-dried and weighed so that results could be expressed in relation to volume, fresh weight or dry weight. The 10 -1 suspension was serially diluted in 0.1% Brij 35 solution to give a range of tenfold dilutions to suit the expected numbers of PFUs in the soil. Usually the dilution range used was from 10 -1 to 10 -3 , but where high numbers were expected, from 10 -2 to 10 -4 . One cm 3 of each of the 3 consecutive dilutions was placed on each of 5 plates of C M A / 2 plus yeast. The 15 plates were placed in an unsealed polythene bag in a 20°C incubator for 1 week and then examined with a binocular dissecting microscope ( x 10) and a phase-contrast microscope ( x 200). The former was usually adequate for observing phaneroplasmodia, but the latter was necessary for observing aphanoplasmodia and protoplasmodia. Incubation was continued for a further 4 weeks in natural daylight on the laboratory bench (18-22°C) with the plates enclosed in a clear plastic seedling-propagating box to minimise drying and risk of contamination, and examined weekly for plasmodia and sporangia. If the plates lost their surface moisture film and appeared dull, sterile distilled water was sprayed onto the agar surface, sufficient to restore the film but not enough to flow freely across the surface. The number of PFUs in the original soil was calculated from the numbers of plasmodium-containing plates at each dilution step with the aid of most-probable-number tables [8,9].

4.2. Tests of the reproducibility of the standard method 2 Experiments were conducted to test the consistency of the standard method in practice. In the first, 9 independent estimations were conducted on a pooled sample of soil. Red marl soil from 4 rectangular cores, 5 x 8 x 3 cm deep, was collected from a field site in grass turf at the edge of an


105 Table 1 Independent estimates by the most-probable-number method of the number of plasmodium-forming units (PFUs) in 9 subsamples of a pooled sample of soil from an apple orchard Sample No.

PFUs. c m - 3 soil

PFUs. g - ] dry soil

1 2 3 4 5 6 7 8 9

800 350 350 1700 450 500 200 1300 500

1720 754 580 2369 698 786 305 2088 841

683

1127

Mean

unsprayed apple orchard at Long Ashton Research Station, Bristol, England (O.S. map reference, ST 535705) and mixed thoroughly. Nine 50-cm3 samples of the mixed soil were separately assayed by the standard procedure, using dilutions of 10 -2 , 10 -3 and 10 -4 . The results are presented in Table 1. The individual estimates ranged from 200-1700 P F U . cm -3 soil, with a mean value of 683 cm -3. The upper and lower 95% confidence limits of a single estimate based on 5 samples from inocula diluted in tenfold steps are 3.3 and 0.303 times the estimate, respectively [10]. The best available estimate of the number of PFUs in this soil is the mean of the 9 separate assays. This lies within the 95% confidence limits of 8 of the 9 individual estimates, and is only fractionally beyond the limit for the single low estimate of 200. The same is true for the data expressed per g dry weight. The method thus furnished essentially reproducible estimates. The second test of consistency was made with soil gathered on a different occasion from the same field site, but instead of a well-mixed fresh collection of soil being divided into sub-samples, an aqueous suspension of soil was divided. 50 cm 3 Soil was suspended in 450 cm 3 sterile 0.1% Brij 35 and 10 samples were taken for independent assays of the number of PFUs present. Because the culture medium and incubation conditions allowed the numbers of certain other organisms to be estimated simultaneously, by the most-probable-

number method, these too were recorded, so that the reproducibility of the estimates of PFUs could be compared with that of estimates of other organisms. 'Presumptive myxoflagellates' were organisms with the distinctive morphology and locomotory behaviour of myxomycete swarm cells. In the absence of evidence that they were able to give rise to plasmodia, we have, for the present, hesitated to use them as a basis for enumerating the abundance of myxomycetes in the soil. Myxobacteria were recognised by their characteristic fruit bodies. The results are presented in Table 2. The 95% confidence limits of 7 of the 10 estimates of PFUs embraced the mean. However, for the other groups of organisms this occurred for all 10 estimates of ciliates, 9 of presumptive myxoflagellates, and 9 of myxobacteria (some of the estimates of amoebae and nematodes were outside the quantifiable range). The results suggest that sometimes the standard method may underestimate the numbers of PFUs present in the soil and there may be a biological reason for this. Firstly, the estimates whose 95% confidence limits did not embrace the mean were the 3 lowest values (170, 250, 250). Secondly, there were 2 results (Tests 2 and 3) for which improbable score sequences were recorded, i.e., 3-3-0 and 4-4-0. When a given dilution of inoculum yields 2 or more positives, the next tenfold higher concentration should yield appreciably more. The occurrence of unlikely most-probable-number results has been discussed by Taylor [12]. Because the results for the other organisms in Table 2 lacked such sequences, the most likely explanation of anomalous results for PFUs is that competition from other organisms in the most concentrated series sometimes led to failure of potential PFUs to give rise to detectable plasmodia. Microscopic examination of isolation plates suggested that ciliates and nematodes were among the deleterious competitors. It may be significant that the 2 anomalous sequences were ones in which the numbers of either ciliates or nematodes were the highest recorded. There is other supporting evidence. In assays by the authors of the abundance of PFUs in artificial microcosms in which only myxomycetes, bacteria and angiosperm roots were present,


106 Table 2 Results of 10 independent estimates by the most-probable-number method of the numbers of plasmodium-forming units (PFUs), presumptive myxoflagellates, amoebae, ciliates, myxobacteria and nematodes in a single sample of soil from an apple orchard Score-sequences a and most-probable-numbers of organisms per cm3 soil

Test No.

PFUs

1 2 3 4 5 6 7 8 9 10

5-4-1 3-3-0 4-4-0 4-3-0 5-4-0 5-4-0 5-4-0 4-3-0 5-3-0 5-5-1

Mean

1700 170 350 250 1300 1300 1300 250 800 3500 1092

Presumptive myxoflagellates

Amoebae

5-4-2 5-5-1 5-5-2 5-5-3 5-5-2 5-5-3 5-5-3 5-5-4 5-5-3 5-5-3

5-5-2 5-5-5 5-5-5 5-5-4 5-5-5 5-5-4 5-5-5 5-5-4 5-5-5 5-5-5

2250 3500 5500 9000 5500 9000 9000 16000 9000 9000

Ciliates 5500 > 18000 > 18000 16000 > 18000 16000 > 18000 16000 > 18000 > 18000

7775

5-2-0 5-4-0 5-2-0 5-2-0 5-3-1 5-2-0 5-2-1 5-2-0 5-2-0 4-2-0

-

500 1300 500 500 1100 500 700 500 500 200

Myxobacteria

Nematodes

5-4-2 5-5-2 5-5-1 5-4-3 5-5-3 5-5-1 5-4-2 5-5-4 5-5-3 5-4-0

2-0-0 0-0-0 2-0-0 1-0-0 1-0-0 0-0-0 1-0-0 0-0-0 1-0-0 0-0-0

630

2250 5500 3500 2750 9000 3500 2250 16000 9000 1300

50 < 20 50 20 20 < 20 20 < 20 20 < 20

5505

a The 'score sequence' is the number of positive plates out of 5 at each of 3 dilutions of the inoculum differing by factors of 10, highest concentration first.

a n o m a l o u s sequences n e v e r arose. Also, in assays in w h i ch soil samples were frozen to destroy the vegetatively active stages o f m y x o m y c e t e s , a n o m a lous sequences were e n c o u n t e r e d only once, possibly because the c o m p e t i n g p o p u l a t i o n s h a d b e e n d e p l e t e d or eliminated. In the course o f a p p l y i n g the s t a n d a r d p r o c e d u r e in studies which will be r e p o r t e d elsewhere, a n o m a l o u s results have p r o v e n to b e u n c o m m o n . In a survey of 38 w h e a t f i e l d soils, only o n e a n o m a l o u s sequence was encountered, a n d in a survey of m o r e t h a n 200 o t h e r soils of diverse types a n d usage there were 4; 3 of t h e m for the same o r c h a r d soil used above. T h e m e t h o d is e v i d e n t l y satisfactory in the m a j o r i t y of soils. Th e o r c h a r d soil used in the a b o v e experim e n t s was the p ri n c ip a l exception, t h o u g h on the o c c a s i o n of the s a m p l i n g date on which the d a t a in T a b l e 1 are based, n o n e of the score sequences was anomalous.

4. 3. Application of the standard enumeration procedure to a wide range of soils T h e foregoing e x p e r i m e n t s a n d similar w o r k i n d i c a t e d that, in local soils, n u m b e r s of P F U s of the o r d er o f 1 0 0 - 1 0 0 0 . c m -3 or g - 1 fresh weight were c o m m o n . T o d e t e r m i n e w h e t h e r these values were representative of soils generally, a n u m b e r of

soils collected f r o m a w i d e r an g e o f geographical situations was assayed. T o ensure s o m e c o m p a r a bility of soil-sampling p r o c e d u r e s in these differen t locations, it was necessary investigate first w h e t h e r the d e p t h f r o m which the sample was t ak en i n f l u e n c e d the result. A 6 - c m - d i a m e t e r core was r e m o v e d in early s u m m e r f r o m the top 8 c m of the soil in the o r c h a r d site specified above. T h e core was d i v i d ed i n t o 2-cm lengths, an d the n u m b e r s of P F U s assayed in each b y the s t a n d a r d procedure. T h e results p r e s e n t e d in T a b l e 3 show that the ab u n d a n c e of P F U s in the s t r a t u m b e t w e e n 6 an d 8 c m

Table 3 Numbers of plasmodium-forming units (PFUs) recovered from each 2-cm length of an 8-cm-long core of soil from an apple orchard Depth of sample (cm)

PFUs. cm-3 of soil *

0-2 2-4 4-6 6-8

5500 a 3500 a 1300 a 250 b

• Estimates with the same letter are not significantly different at the P = 5% level.


107 Table 4 Abundance of plasmodium-forming units in soil samples from geographically diverse locations Location

Habitat

AFRICA Zimbabwe Harare, Nat. Bot. Gdns. Harare, Greenwood Pk. Sabi-Limpopo Valley

Kopje area Lawns Lowveld tree savanna

20 110 110

Acacia confusa woodland Mud Regenerating slope after felling Rubber plantation Rubber plantation Slope understorey Slope understorey

<2* 17 4 110 80 4 11

Hong Kong New Territories New Territories New Territories

Broadleaf evergreen forest Herbaceous cover by path Pine wood

130 < 20 < 20

AUSTRALASIA Australia Gawler Ranges, S. Aust. Gawler Ranges, S. Aust. Gawler Ranges, S. Aust.

Hummock grassland hillside Open Acacia sowdeni woodland Barren plain

EUROPE Albania Butrint Fier Lezhe Saranda Saranda Shkodra

Low grass Low grass hill vegetation Woodland litter Lush meadow Lush meadow Garden soil

ASIA China Hainan Hainan Hainan Hainan Hainan Hainan Hainan

Germany Mosbach, Baden-Wfirttemberg Mosbach, Baden-Wiirttemberg Mosbach, Baden-Wilrttemberg Mosbach, Baden-Wiirttemberg Mosbach, Baden-Wiirttemberg Mosbaeh, Baden-Wiirttemberg Mosbach, Baden-Wtirttemberg Walldurn, Baden-WOrttemberg Walldurn, Baden-Wiirttemberg Walldurn, Baden-Wilrttemberg Walldurn, Baden-W~ttemberg Switzerland Gstaad Rinderberg Peak NORTH AMERICA U.S.A. Bryce Canyon, UT Death Valley (Furnace Creek), CA

Beech wood Grass ley Kale field Lawn wormcasts Mangold field Ploughed stubble Stubble Beech wood Conifer wood Hornbeam wood Oak wood Meadowland Meadow above treeline, 2500 m

PFUs. g - 1 fresh wt.

14 5 5

170 11 > 1800 550 65 25 < 20 5500 200 50 9000 350 140 < 20 < 20 50 40 13 17

Scattered pines on sandy soil

2

Almost no vegetation

2


108 Table 4 (continued) Location Grand Canyon (North Rim), AZ Grand Canyon (South Rim), AZ Inyo National Forest, CA Las Vegas, NV Painted Desert, AZ Sonoran Desert, hr. Phoenix, AZ Yosemite National Park, CA

Habitat

P F U s . g - ~ fresh wt.

Pines on brown earth Pines on sandy soil Coniferous forest Roadside desert Desert flora Desert flora Coniferous forest

8 14 5 2 < 2 35 < 2

* The symbol < signifies that if myxomycetes were present they were below the specified limit of detection, none being recovered.

deep was significantly less ( P ~< 5%) than in the upper layers of the soil. Though differences between the estimates for the 3 upper layers were not statistically significant, the numbers suggest a steady decline with depth. (The ratios of 2 separate estimates obtained by the described method, which may be judged as significantly different at different critical values of probability for 2-tailed tests, can be calculated [10]; these are 3.73 for P = 10%, 4.80 for P = 5%, 7.87 for P = 1% and 13.95 for P = 0.1%). The observed vertical distribution of myxomycetes resembles that of protozoa in temperate soils [15,16]. In view of these results, those who agreed to collect soil samples were therefore asked to take them from the first 4 cm of the soil profile. The samples of soil in field condition were placed in clean plastic containers and assayed within 4 weeks of collection, usually within 2. In other experiments, samples were assayed on the day that they were collected, but in order to establish a general indication of the abundance of myxomycetes in soils worldwide, this practice was waived. The results of applying the standard enumeration procedure to these samples are given in Table 4. The data suggest that myxomycetes are of widespread distribution and that their abundance is sometimes high. In only 8 of the 44 soils assayed were no plasmodia recovered, and even these may have been soils with numbers of PFUs below the limits of detection of the standard method. It may be ecologically significant that 6 of these 8 nonyielding soils were from woodland. On the other hand, one assay of an Albanian woodland soil revealed a high abundance. Four of the desert soils

yielded myxomycetes, a result which accords with floristic studies which show that myxomycetes can be found in desert regions [17]. In general, higher numbers were recovered from grassland and agricultural soils than from woodland soils. In view of these data and those in the preceding tables it appears that the role of myxomycetes in terrestrial ecology has been underestimated in the past. 4.4. General discussion The question of the nature of the enumerated PFUs has yet to be resolved. A PFU might be a spore, swarm cell, myxamoeba, microcyst, or part of a plasmodium or sclerotium. The evidence we have is compatible with PFUs in the soil being uninucleate stages rather than plasmodia, but the whole question of the form of activity of myxomycetes in the soil requires further examination. Chuang and Ko [18] present an equation which relates propagule size to the maximum population density of the microorganisms within the soil, and which may be used predictively. If it is assumed that swarm cells, myxamoebae and microcysts are the likely form in which myxomycetes occur in the soil, then, on the supposition, based on observation, that their volumes will be mostly between 100-400 /~m3, the corresponding population densities can be calculated as being between 4800-2270 g-1 of soil, respectively. Such predictions are of the order of the numbers of PFUs reported here in a number of soils, and suggest that such uninucleate forms may be the dominant state in which myxomycetes live in soil, at least when present in numbers in the order of thousands per g.


109 It is likely that the enumeration m e t h o d described underestimates the numbers of P F U s present in the soil, because in at least some m y x o m y cetes, single spores, amoebae or microcysts are incapable of yielding plasmodia. Instead, 2 compatible cells are required for plasmodium formation. This situation could lead to a several-fold underestimation. It m a y therefore be significant that the 'presumptive myxoflagellates' in Table 2, whose m o r p h o l o g y and m o v e m e n t closely resembled myxomycete swarm cells, were present in numbers about sevenfold higher than the PFUs. It is possible that all or most of these presumptive myxoflagellaes genuinely were myxomycete cells. However, until the p o p u l a t i o n structure of myxomycetes in soil samples has been studied, it is prudent to base conclusions on the numbers of PFUs, which are unequivocally m y x o m y c e t o u s even if they are underestimates. The majority of myxomycetes recovered from British soils and induced to fruit proved to be Didymium species. Whether this reflects selectivity of the enumeration method, or the capacity of these Didymium species for rapid growth, or truly reflects the a b u n d a n c e of D i d y m i a in the soil microflora, is the subject of continuing investigation. It should be noted that species of other genera have been successfully isolated on the m e d i u m used.

ACKNOWLEDGEMENTS During the period in which most of the research reported here was conducted, A.F. was supported b y a Science and Engineering Research Council studentship. Part of the investigation was supported by a grant to M.F.M. from the Natural Environment Research Council. W e thank colleagues who furnished us with soil samples from diverse localities.

REFERENCES [1] Madelin, M.F. (1984) Myxomycetes, microorganisms and animals: a model of diversity in animal-microbial interactions, in Invertebrate-Microbial Interactions (Anderson, J.M., Rayner, A.D.M. and Walton, D.W.H., Eds) pp. 1-33, Cambridge University Press, London. [2] Madelin, M.F. (1984) Myxomycete data of ecological significance, Trans. Br. Mycol. SOc. 83, 1-19 [3] Farquharson, C.O. and Lister, G. (1916) Notes on South Nigerian Mycetozoa, J. Bot. 54, 121-133. [4] Indira, P.U. (1968) Some slime moulds from Southern India, IX, J. Indian Bot. 41, 792-799. [5] Lister, G. (1918) The Mycetozoa: a short history of their study in Britain; an account of their habits generally; and a list of species recorded, Essex Field Club Memoirs 6, 1-54. [6] Thorn, C. and Raper, K.B. (1930) Myxamoebae in soil and decomposing crop residues, J. Wash. Acad. Sci. 20, 362-370. [7] Warcup, J.H. (1950) The soil plate method for the isolation of fungi from soft, Nature 166, 117-118. [8] Alexander, M. (1965) Most-probable-number method for microbial populations, in Methods in Soil Analysis, Part 2. Chemical and Microbial Properties (Black, C.A., Ed.) pp. 1467-1472, Am. SOc.Agronomy, Madison, WI. [9] Ministry of Health; Ministry of Housing and Local Government (1969) The bacteriological examination of water supplies, in Reports on Public Health and Medical Subjects No. 71, 4th ed. H.M. Stationery Office, London. [10] Cochran, W.G. (1950) Estimation of bacterial densities by means of the 'most probable number', Biometrics 6, 105-116. [11] Maloy, O.C. and Alexander, M. (1958) The 'most probable number' method for estimating populations of plant pathogenic organisms in the soil, Phytopathology 48, 126-128. [12] Taylor, J. (1962) The estimation of numbers of bacteria by tenfold dilution series, J. Appl. Bacteriol. 25, 54-61. [13] Hok, K.A. (1954) Studies of the nutrition of myxomycete plasmodia, Am. J. Bot. 41, 792-799. [14] Gray, W.D. and Alexopoulos, C.J. (1968) The Biology of Myxomycetes. Ronald Press, New York. [15] Stout, J.D. and Heal, O.W. (1967) Protozoa, in Soil Biology (Burgess, A. and Raw, F., Eds.). pp. 149-195, Academic Press, New York. [16] Sleigh, M.A. (1973) The Biology of Protozoa, Edward Arnold, London. [17] Blackwell, M. and Gilbertson, R.L. (1980) Sonoran Desert Myxomycetes, Myeotaxon 11, 139-149. [18] Chuang, T.Y. and Ko, W.H. (1981) Propagule size: its relation to population density of microorganisms in soil, Soil Biol. Biochem. 13, 185-190.


A METHOD FOR THE ENUMERATION OF MYXOMYCETES IN SOILS AND ITS

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