Microb Ecol (1993) 25:83-92 CROBIAL ECOLOGY © 1993 Springer-Verlag New York Inc.

Microfungal Species Composition and Fungal Biomass in a Coniferous Forest Soil Polluted by Alkaline Deposition

Hannu Fritze 1 and Erland B~th 2 ~Department of Forest Ecology, Finnish Forest Research Institute, P.O. Box 18, SF-01301 Vantaa, Finland; and 2Department of Microbial Ecology, Lund University, Helgonav~igen 5, S-22362 Lund, Sweden

Received: June 2, 1992; Revised: September 1, 1992

Abstract. Isolations of soil microfungi from the humus (F/H-layer) of a coniferous forest soil which was either unpolluted (pH 4.1) or polluted (pH 6.6) for 25 years by deposition of alkaline dust, were made by soil washing and spore plating. Both techniques revealed similar changes in species composi- tion. Alkaline dust exposure caused a reduction in overall species numbers, but led to higher relative isolation frequencies of Mortierella alpina, Oidiodendron tenuissimum, Penicillium montanese, Sagenomella verticillata, and Trichos- poriella sporotrichioides. The incidence of M. isabellina, O. cf. clamydos- porium, P. spinulosum, Penicillium sp. 1, P. sclerotiorum, Trichoderma vir- ide, and Verticillium bulbillosum was reduced on polluted sites. The amount of the mainly fungal-derived phospholipid fatty acid 18 : 2~o6 decreased by 23%, while the amount of ergosterol increased by 9% in the polluted soil.

Introduction

Investigations of microbial activity, biomass, and species composition are often made in soils around point emission sources in order to assess the environmental impact of the resulting pollution. To date, the response to heavy metals has received most attention [4, 10] although the effects of sulphur dioxide and elemental sulphur [6, 23] and the deposition emanating from an oil refinery [12] have also been studied. Recently, the effect of alkaline dust emissions from an iron and steel works on forest soil microbial activity and biomass was studied by Fritze [11]. As a result of deposition of the dust, the bulk soil pH of the forest humus layer increased from 4.1 in unpolluted soils, to about 6.6 near the factory. The change was associated with enhanced soil respiration, but not with a higher mass loss in needle litter after a one year soil incubation period. Furthermore, the total length of fungal hyphae nearly doubled in the polluted soils, although there was only a small change in microbial biomass C and N. The soil microbial biomass either decreased or in-

Offprint requests to: H. Fritze.

84 H. Fritze and E. B~hth

creased slightly when expressed per soil volume or organic matter (loss-on-igni- tion), respectively [11].

The purpose of this study was to further investigate the effects of alkaline deposition with special reference to changes in the soil microfungal species compo- sition. Two different isolation techniques, soil washing and spore plating, were compared. Since total length of fungal hyphae includes both living and dead hyphae, ergosterol and the phospholipid fatty acid 18 : 2eo6 were quantified in order to estimate viable fungal biomass. The results allow comparison with liming experiments of coniferous forest soils, because the study sites were effectively exposed to large overdoses of lime as a result of the use of limestone in steel processing.

Methods

Site Description and Soil Sampling

The Koverhar iron and steel works was established in 1961 and is situated in Southern Finland on the Baltic sea coast. Twenty of the 24 previously established sites located in a dry Calluna type Scots pine (Pinus sylvestris L.) forest [11] were sampled in September 1991. Half of these sites were exposed to alkaline dust, while the remaining ten sites served as controls. Ten individual soil core samples (F/H-layer) were obtained from each study site, and the resulting twenty bulk soil samples were sieved (mesh size, 4 mm) and visible plant material removed before storage at 4°C. For the determination of the organic matter content as loss-on-ignition (LOI), oven dried (105°C) samples were heated to 550°C for a minimum of 4 hours.

Fungal Isolation

Six randomly chosen control and polluted bulk soil samples were subjected to two isolation procedures. A modified soil washing technique [3] was used to produce soil particles (180 from each site) sized between 80 and 100 Ixm. These were placed on 1% malt-extract agar (MA) plates containing 50 mg liter- ~ chlortetracycline and maintained at room temperature (average 22°C). Fast growing fungi were transferred to new MA plates after 3 days, and a second survey of the plates was made after 3 weeks, when the slower growing fungi had grown out from other particles. The relative frequency of isolation of each species was calculated as a percentage of the total isolation number for each site.

Colonies produced mostly from spores were isolated after homogenizing 10 g wet weight (ww) of soil with 100 ml sterile water for 2 rain in a Sorvall Omnimixer at 80% of the maximum rated speed, followed by filtration through a 50 p,m sieve. Successive dilutions were then plated on MA and incubated at 15°C for ten days. One hundred colonies from plates containing 20 colonies or less were isolated from each site.

Fungal Biomass

All twenty samples were subjected to the two fungal biomass measurements. The phospholipid fatty acid 18 : 20)6 was quantified as follows. Fresh soil (0.50 g ww) was extracted with a one-phase mixture consisting of chloroform, methanol, and citrate buffer (0.15 M, pH 4.0) in the proportions 1 : 2 : 0.8 (v/v/v) for at least 2 h [13, modified after 8]. The soil was removed by centrifugation, and the supernatant split into two phases by addition of chloroform and citrate buffer. The lipid material in the lower phase was fractionated into neutral, glyco-, and polar lipids (phospholipids) on silicic acid columns. After addition of methyl nonadecanoate as an internal standard to the phospholipid fraction,

Soil Microfuugi and Alkaline Deposition 85

each sample was subjected to a mild alkaline methanolysis. The resulting fatty acid methyl esters were separated on a Hewlett Packard 5890 gas chromatograph equipped with a flame ionization detector and a 50 m HP5 capillary column,

Ergosterol was extracted and quantified as follows [ 15]. Fresh soil (0,50 g ww), with 7-dehydrocho- lesterol added (100 p.1 of a 0.91 p~g p.1-1 solution in methanol) as an internal standard, was extracted with 2 ml of methanol, vortexed, and centrifuged for 10 rain at 3000 rpm. The supernatant was removed and the remaining soil washed twice more with the same volume of methanol. To the combined methanol supernatants, 0.2 ml 4% KOH (in 96% ethanol) per ml of methanol was added, and the solution was incubated for 30 min at 80°C. Distilled water and hexane (2 ml each) were then added and the hexane phase separated. After a second hexane extraction, the combined hexane phases were dried under nitrogen. The. extracted sterol material was dissolved in 500 p~l acetonitrile (CH3CN; Fissons FSA supplies, Sigma) and analysed by HPLC. The HPLC was equipped with a Nova Pak C 18 reverse-phase column, and ergosterol was detected at 282 nm. Hexane-isopropanol-acetonitrile (5 : 5 : 90) was used as a carrier. All chemical solvents were of HPLC grade.

Statistical Analysis

An analysis of variance (ANOVA) was used to test differences between polluted and control sites as to biomass and the number of spores in the soil. The data were normally distributed except for the spores, which required a cosine transformation. The Mann-Whitney U-test was used for the fungal species frequency data. The Statistix 3.1 program (Analytical Software) was used for statistical testing. The fungal species data were also subjected to a principal component analysis, including the most com- monly isolated taxa from both isolation techniques. The Sirius program [ 18] was used for the analysis.

Results

With the soil washing technique, the species with the highest isolat ion f requency are l is ted in Table 1. The control soils had a much r icher Penicillium flora. In addi t ion to the species recorded in Table 1, Penicillium thomii Maire , P. lividum West l ing , P. loliense Pitt, and P. soppii Zaleski were also isolated, but only P . soppii was found in the pol lu ted sites. The only Peniciltium i sola ted in much higher f requencies f rom the pol lu ted sites was P. montanense, while P. spinulosum, P. cf brevi-compactum, Penicillium sp. 1 (an unident i f ied monover t ic i l la te species with rough conidia) , and P. sclerotigenum were more c o m m o n in the control sites. Seven different species of Oidiodendron were isolated. O. griseum and O. echinu- latum did not show any s ignif icant differences in abundance be tween the pol lu ted and control sites, whereas O. tenuissimum and O. cf. clamydosporium were iso- l a ted in much higher numbers f rom pol lu ted and control soil par t ic les , respect ive ly . O. truncatum Barron and O. rhodogenum Robak were found only in low numbers in the control sites, and O. scytaloides was restr icted to the pol lu ted soils. The two Verticillium species showed clear di f ferences in pol lu t ion response. V. lecanii was isola ted in h igher numbers f rom the pol lu ted soils, whereas V. bulbillosum showed an ins ignif icant b u t h igher isolat ion f requency in the control soils. Mortierella alpina was the only Mortierella i sola ted f rom the pol lu ted sites and this species was not found in the controls , whereas M. ramanniana (M611er) Linnemann, M. isabel- lina, M. verticillata Linnemann, and M. macrocystis W . Gains were isolated f rom the control soils. Sagenomella verticillata and S. grisiovirides (Onions & Barron) W. Gams were obta ined only f rom the pol lu ted sites. The highest isolat ion frequen-

86 H. Fritze and E. B~hth

Table 1. Fungal isolation frequencies (% total no. isolates) of the most abundant species isolated using the soil washing and spore plating methods

Soil washing Spore plating

Control Polluted Control Polluted

Penicillium spinulosum Thorn 3.6 0.3 4.0 P. montanense Christensen & Backus 4.9 12.3 a 7.7 P. cf brevi-eompactum Dierckx 1.8 0.0 ~ 3.2 Penicillium. sp.1 2.9 0.2 a 4.8 P. sclerotiorum van Beyma 1.4 0.0 6.8 Oidiodendron griseum Robak 4.3 3.8 11.8 O. tenuissimum (Peck) Hughes 1.2 10.6 a 0.3 O. echinulatum Bah'on 4.5 3.2 2.3 O. cf ¢hlamydosporicum Morrall 10.4 0.0 a 10.6 O. scytaloides W. Gains & S6derstr6m 0.0 0.3 0.0 Trichoderma viride Pers.:Fr. 1.9 0.0 ~ 0.2 Verticillium lecanii (Zimm.) Vi6gas 1.8 6.6 a 1.3 Verticillium bulbillosum W. Gams & Malla 1.0 0.2 1.7 MortiereUa isabellina Oudem. 1.2 0.0 a 2.0 Mortierella alpina Peyron. 0.0 1.5 a 0.0 Sagenomella verticillata W. Gams & S6derstr6m 0.0 1.9 a 0.0 Trichosporiella sporotrichioides van Oorschot 0.4 0.0 1.5 Trichosporiella sp. 1 0.0 0.0 0.7 Yeast 1.4 6.8 ~ 12.1

0 . 2 a

15.0 ~ 3.8 0.0 a 0 . 2 a

0.5 a 22.0 a 2.8 1 . 0 a

2.0 a 0.0 0.5 0.3 ~ 0.0 ~ 6.2 a 5.2 ~ 5.0 a 2.5

13.6

aSignificant difference from the control (P < 0.05, Mann-Whitney U-test)

Table 2. Effect of the alkaline dust pollution on the fungal colonization index (isolates per plated soil particle) from soil washing, on number of fungal spores from spore plating, and on the amounts of the phospholipid fatty acid 18:2to6 and erogosterol. Means -+ S.E.

Control Polluted

Colonization index 0.50 -+ 0.02 0.37 -+ 0.02 a Spores (No g-1 LOI x 105) 9.1 +- 0.6 10.5 + 2.2 18:2~o6 (nmol g 1 LOI) 465 --- 34 360 -+ 16 a Ergosterol (p~g g-1 LOI) 232 -+ 5 253 -+ 21

aMeans are significantly different (P < 0.05, ANOVA)

cies were for non-sporulat ing fungi, which comprised 46.5 % and 45.0% of the total fungal isolates in the control and polluted sites, respectively.

From the total of 920 isolations using the soil washing technique, 37 and 22 different microfungal taxa were identified from the control and the polluted sites, respectively. It was possible to isolate more fungi originating from soil particles in the control sites than from the polluted sites, as indicated from the higher coloniza- t ion index (Table 2). Fifty percent of the control soil particles were associated with a fungus, as compared to only 37% in the polluted soil.

From spore plating, 1200 isolations were made resulting in the detection of 37 and 35 different fungal taxa in the control and polluted soil, respectively. The species with the highest isolation frequencies are listed in Table 1. The changes in fungal species composit ion in the polluted soils were similar to those detected by

Soil Microfungi and Alkaline Deposition 87

soil washing. For a given species, similar results from the two methods were usually observed when single sites were considered. Differences relating to the soil washing technique were observed in the isolation frequency ofP. cf. brevi-compac- turn, O. griseum, and V. lecanii. No relationship between pollution and P. cf. brevi-compactum was observed, and the latter two fungi were isolated in higher numbers from the control soils. Two species of Trichosporiella, T. sporotrichio- ides, and an unidentified species called Trichosporiella sp. 1, were more frequently isolated using spore plating, and both fungi showed a higher isolation frequency in the polluted soils. No pollution-dependent differences in the amount of yeast were found when using the spore plating technique, as compared to soil washing.

Other taxa isolated from control and/or polluted sites in low numbers and not mentioned above were Absidia glauca Hagem, Acremonium butyri (van Beyma) W. Gams, A. cerealis (Karst) W. Gams, Aphanocladium album (Preuss) W. Gams, Beauveria bassiana Balsamo (Vuill.), Chaunopycnis alba W. Gams, Geomyces pannorum (Link) Sigler & Carmichael, Mortierella alliacea Linnemann, M. parvispora Linnemann, M. pulchella Linnemann, Mucor silvaticus Hagem, Oidio- dendron cf. pilicola Y. Kobayasi, Paecilomyces farinosus (Holm: Fr.) A.H. Brown & G. Smith, Penicillium citrinum Thorn, P. lagena (Delitsch) Stolk & Samson, Phialophora sp., Sagenemonella striatospora (Onions & Barron) W. Gains, Thysanophora penicillioides (Roum.) Kendrick, Tolypocladium niveum (Rostrup) Bissett, and Torula herbaum Link : Fr. In soils subjected to both extrac- tion methods, a total of 47 and 35 different taxa were isolated from the control and polluted areas, respectively.

The fungal data from Table 1 (for both soil washing and spore plating) were subjected to a PC-analysis, which from the first principal component resulted in a complete separation of the polluted and control sites (Fig. 1). The first principal component thus represented the impact of the pollution and accounted for 31% of the variation of the fungal isolation data. The second principal component (5% of the variation) did not correlate with the pollution gradient.

The number of spores present in the soil did not differ between the treatments, being 9.1 × 105 g-1 LOI for the control and 10.5 x 105 g-1 LOI for the polluted area (Table 2). The viable fungal biomass estimations, based on the amount of the phospholipid fatty acid 18 : 20)6 and ergosterol, are presented in Table 2. A 23% reduction in the amount of this fatty acid in the polluted area contrasted with a 9% increase in ergosterol in the same area.

Discussion

The species benefitting most from the alkaline deposition were P. montanense, O. tennuissimum, O. scytaloides, V. lecanii, M. alpina, S. verticillata, T. sporotri- chioides, Trichospoirella sp. 1, and yeast M. alpina was the only Mortierella species recorded in polluted soil; this species has been commonly reported in neutral and alkaline habitats [7, 9, 16], and may be an indicator species for raised soil pH.

O. scytaloides is generally found in the mineral soil layers of coniferous soil [14, 21], and S. verticillata has also been isolated frequently from the A 2 and B layers of a clear-cut pine forest [2]. Since the mineral soil layer generally has a higher pH

88 H. Fritze and E. Bh~tth

IX P

P

P

P

P

P

+ C

C

C

C

6

PC1 Fig. 1. Principal component analysis of the most commonly isolated microfungi in the Koverhar area using both data from the soil washing and the spore counting techniques. C = non-polluted control sites, P = alkaline dust polluted sites. The first (PC1) and second (PC2) principal component account for 31% and .5% of the variation in the microfungal data, respectively.

than the humus layer, the increased isolation frequency of O. scytaloides and S. verticillata would seem to reflect a general increase to more neutral pH values. The deposition of the mineral dust onto the forest floor, which causes the humus layer to resemble a rnore mineralic soil [11], could also be the reason for the higher isolation frequency of these two species. This could also be the case for M. alpina, which has been isolated more frequently from the mineral B-horizon than from the other soil horizons [21]~

Species which clearly decreased in the polluted sites were P. spinulosum, Peni- cillium sp: 1, O. cf. elamydosporium,T, viride, and all Mortierella species except M. alpina. P. spinulosum, T. viride, and O. echinulatum are reported to increase in

Soil Microfungi and Alkaline Deposition 89

coniferous soil after artificial acidification [5] and fertilization with NH4NO 3 [1]. Both these treatments decrease the soil pH at least temporarily. In our study, using the soil washing technique, these three fungi were isolated in higher frequencies in the acidic control sites. However, for O. echinulatum the difference between the neutral and acidic sites was not statistically significant, and similar numbers of conidia were detected in all the sites.

Microfungi have been isolated from coniferous soils with the soil washing technique after liming and wood ash treatment of the forest [Bfifith E, Arnebrant K, in press, Biol Fertil Soils]. Liming decreased the abundance of P. spinulosum, O. cf. truncatum, and Mortierella spp., while the isolation frequencies of P. cf. brevi-compactum, Trichoderma polysporum (Link : Fr.) Rifai, and T. sporotri- chioides were increased. The results are similar to ours with respect to P. spinulo- sum, Mortierella spp., and T. sporotrichioides. However, reduced numbers of P. cf. brevi-compactum were found in our polluted area, particularly when assessed by soil washing, although slightly higher numbers were found with spore plating. T. polysporum was never isolated from our study area.

The reduced species richness in polluted sites was evident both in the total number of taxa and also within the different genera. For example, eight species of Mortierella were found in the control sites, but only one in the polluted sites. The change was attributable to several species common in this type of forest soil [21] disappearing from the polluted soils. Except for Trichosporiella sp. 1, no fungal species were found in the polluted sites that had not been previously identified in acidic, unpolluted forest soil. Fungi that are characteristic for agricultural soil with a neutral pH, like Cylindrocarpon, Fusarium, and Michrodochium, were not de- tected in the polluted area. This indicates that, for these fungal genera, other factors in addition to the low soil pH determine their absence from forest soils. In a study of the microflora of limed and reclaimed peat bogs [17], Fusarium oxysporum was found in the peat of the reclaimed area with the highest pH (7.5), but not in the virgin bog containing acidic peat (pH 3.8). However, the reclaimed neutral peat area had been used for vegetable crop production over an 11 year period, while in the Koverhar area, which included our study sites, the coniferous forest still prevails.

Fungal biomass can be assessed in soil by measuring chitin; ergosterol, or the mainly fungal phospholipid fatty acid 18 : 2o~6 [22]. The amounts of the two latter compounds are probably better indicators of the living fungal biomass, since they form part of the cell membrane. However, linoleic acid (18 : 2eo6) is also known to occur as a dominant fraction of all fatty acids in Pinus sylvestris [20], and so must be assumed to be rapidly decomposed from this source, if the results are to be interpretable. Increased total hyphal length, when expessed on either a soil volume or organic matter (LOI) basis, in the area receiving alkaline deposition was reported by Fritze [11]. The present study revealed a lower fungal biomass when estimated as the amount of 18 : 2~o6, but a higher biomass in the polluted soil when measured with the ergosterol method (Table 2), so no clear influence of the alkaline deposi- tion on the living fungal biomass could be found. Additional evidence for an unaffected fungal biomass can be seen from the lack ofpH effects on spore numbers (Table 2). Furthermore, although the colonization index from the soil washings differed between polluted and control sites, this difference disappears if one as- sumes that the difference in LOI between the two treatments was found in the soil

90 H. Fritze and E. B~tth

Table 3. Ratio of isolation frequencies of the spore plating and soil washing method in the control and polluted area

Control Polluted

PeniciUium spinulosum 1.1 0.6 P. montanense 1.6 1.2 P. cf brevi-compactum 1.8 high ~ Penicillium. sp. 1 1.7 0.0 P. sclerotiorum 4.8 high Oidiodendron griseum 2.8 0.13 b O. tenuissimum 0.3 2. I b O. echinulatum 0.5 0.9 O. cf chlamydosporicum 1.2 high O. scytaloides ~ 7.4 Trichoderma viride 0.08 - - Verticillium lecanii 0.7 0.07 Verticillium bulbillosum 1.7 1.4 Mortierella isabellina 1.7 - - Mortierella alpina - - 4.0 Sagenomella verticillata - - 2.7 Trichosporiella sporotrichioides 3.9 high Trichosporiella sp. 1 high high Yeast 8.4 2.0 b

a Probably high ratio since no species were recorded with the soil washing technique bSignificant difference from the control (P < 0.05, Mann-Whitney U-test) c No isolates recorded

particles used (microscopical observation) and that not many fungi were isolated from non-organic particles.

The discrepancy between our fungal biomass estimations and the length of the fungal hyphae in the same area [11] could be due to the fact that the latter was determined from microscopial measurements, which would include both living and dead mycelium. This could mean that the soil of the polluted area contained many non-viable hyphae with low decomposition rates. However, no differences in the decomposition rate of needle litter were found among the various sites [11].

Few comparisons between the soil washing and the plate count techniques (similar to our spore coun0 have been made since the development of the former method [19]. Our study confirmed that heavily sporulating fungi are favored by plating techniques, while soil washing enables isolation of many non-sporulating fungi. We found about 45% nonsporulating fungi using soil washing, but almost no such isolates using spore plating.

By dividing the isolation frequencies obtained from spore plating by those from soil washing for individual species in Table 1, it is possible to assess the selective influence of the different isolation method (Table 3, control area). Thus, Penici l - l ium were usually more common using spore plating, while the opposite result was found for Trichoderma. Similar results were reported by B ~ t h [3]. Differences within a genus (e.g., Oidiodendron and Penic i l l ium) were also evident. Both we and Bhhth [3] found, for example, that the isolation of P. sclerotigenum and to some

Soil Microfungi and Alkaline Deposition 91

extent P. montanense was very much favored by plate count techniques, while P. spinulosum, for example, was equally common using either isolation technique.

Soil washing is supposed to favor the isolation of fungi from hyphae in the soil, and thus might reflect the hyphal length, that is the biomass, of the fungi. One reason for the different isolation frequencies for the two methods could thus be that different species have different capacities to produce spores per biomass unit, and the calculated ratio (spore plating/soil washing) could be an index of "sporulation capacity" or spore survival rates. However, it could also be due to different washing efficiencies for different spores. Wet conidia, like those of Trichoderma, are probably more easily washed away from particles than dry conidia produced by P enicillium and Oidiodendron.

Although it is difficult to compare "sporulation capacity" for different species, due to the possibility of differential washing efficiency for different types of spores, it should be possible to compare polluted and unpolluted sites in this respect, assuming that the washing efficiency was similar for all sites. On this basis, pollution lowered the "sporulation capacity" of P. spinulosum, Penicillium sp. 1, O. griseum, and V. lecanii, while the "sporulation capacity" was higher in the polluted sites for P. cf. brevi-compactum, O. tenuissimum, and T. sporotrichioides (Table 3, not significant in all cases). The "sporulation capacity" ofP. montanense, O. echinulatum, and V. bulbillosum was unaffected.

Two hypotheses could account for altered "sporulation capacity". If a fungus is stressed by a more hostile environment, production of more spores per biomass unit would enhance the chances of survival through dormancy or spread of spores to more suitable environments. However, if the environment becomes very unsuit- able, fewer spores would be produced. Both effects have been found in pure culture studies where heavy metal was the stress agent (K. Amebrant, pers. comm.). Thus, in the former case, one would expect species that are less often found in polluted sites to have a higher "sporulation capacity" in these sites compared to control sites and vice versa for species that most often are isolated from polluted soils. For the second hypothesis, the opposite will be the case. The second hypothesis appeared to be true for P. spinulosum, Penicillium sp., 1., O. griseum, O. tenuissimum, and T. sporotrichioides, while it was not true for P cf brevi-compactum. While the uncertainty of whether it is "sporulation capacity" or the spore life-span that is affected by alkaline deposition should be borne in mind, this work represents the first attempt to use a comparison of the two isolation methods in this way.

Similar results concerning microfungal species composition were found with both isolation techniques. Therefore, although soil washing may give a more realistic picture of the soil microfungal flora compared to spore plating, both methods may be equally sensitive for detecting changes due to pollution, manage- ment practices, environmental disturbances, etc. Since spore plating is faster, it might be a better choice when the primary aim of an investigation is only the detection of differences due to treatments.

Acknowledgments. We thank A. Frosteg~rd, K. Arnebrant, and A.-M. Fransson for their valuable help. The work was supported by a grant from the Federal European Microbiological Society (FEMS) to H. F. and the English was corrected by Robin Sen.

92 H. Fritze and E. Bfi~th

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