Freshu'ater Biology (1992) 28, 1-7

Effects of drying and freezing autumn leaves on leachingand colonization by aquatic hyphomycetes

FELIX B A R L O C H E RDepartment of Biology, Mount Allison University, Sackville, NB, EOA 3C0. Canada

SUMMARYI

1. Drying or freezing autumn-shed leaves of Betula papyrifera, Ulmus americana and Acersaccharum increased the leaching of soluble substances. The difference between freshand treated leaves was most pronounced in birch.2. Dried and frozen leaves supported more spedes of aquatic hyphomycetes during earlydecay, and conidium production from these fungi was higher.3. Anguillospora filiformis was the most prolific spore producer on most samples. It declinedin later stages of decay, when Articulospora tetradadia became more common. CrucdlasubtiJis, an unspecialized parasite of aquatic hyphomycetes, was quite common in manysamples.4. In the autumn of 1990, 50% of elm and maple leaves, and 30% of birch leaves wereshed before the air temperature dropped below 0°C; 10% (elm, maple) to 30% (birch)experienced more than one freezing period before being shed.5. Water-soluble extracts from leaves inhibited radial growth of four fungal spedes.Maple extracts generally had the greatest inhibitory effect, followed by birch and elm.

Introduction

The decomposition of autumn-shed leaves in streamsis believed to be initiated by leaching, a rapid abioticremoval of amino acids, carbohydrates, phenolicsand other soluble substances. It leads to a loss of upto 30% of the original weight of the leaf within 24-48 h of its immersion (Cummins, 1974; Suberkropp,Godshalk & Klug, 1976; Webster & Benfietd, 1986).Gessner & Schwoerbel (1989) and Gessner, Meyer &Schwoerbel (1991) recently reported that no such lossoccurs when fresh, rather than predried, alder andwillow leaves were used. Fungal colonization pro-ceeded more quickly on dried than on fresh alderleaves (Barlocher, 1991), presumably because thesoluble substances that are retained in fresh leavesinhibit fungal growth (Barlocher, 1990). Exposureof fresh alder leaves to freezing temperatures alsoincreased the rate of leaching and fungal colonization(Barlocher, 1991). The aim of the present study wasto extend these observations to leaves of three ad-ditional trees, namely paper birch {Betula papyrifera

Marsh.), American elm (Ulmus americana L.) andsugar maple [Acer saccharum Marsh.). The effect ofdrying and freezing on weight loss due to leachingand on colonization by aquatic hyphomycetes wereexamined. In addition, aqueous extracts of the threeleaf species were tested for their ability to inhibitfunga! growth.

Materials and Methods

All leaves were collected from three individual trees(Betula papyrifera, Ulmus americana, Acer saccharum)

by gently shaking branches and collecting freshlyshed leaves. No attempt was made to select unblem-ished leaves. As a result, variable proportions of theoriginal leaf area had been lost due to insect attackand other damage. The loss was estimated to rangefrom 1 to 5% in birch, 5 to 10% in maple, and 30 to50% in elm leaves. Damaged areas were generallysurrounded by a thin border of dry, brown tissue.

Leaves were dried at 20°C for 7 days (dried leaves);exposed to — 12°C for 24 h (frozen leaves) or used

2 F. Barlocher

within 3h of collection (fresh leaves). Subsamplesof fresh, dried, or frozen leaves were dried at 105°Cfor 24 h to determine dry weight.

To determine weight loss due to initial leaching,twenty fresh, dried or frozen leaves were weighedindividually, placed in 250-ml Erlenmeyer flasks filledwith 100 ml distilled sterilized water, and put on ashaker (100 rpm; 5°C) for 72 h. Leaves were thendried (lOS C) and weighed, and weight loss due toleaching was calculated.

To prepare aqueous extracts, ground-up leaf material(passed through a 250-nm steel mesh) was shaken insterile distilled water (lOg per litre, 4°C, 50rpm).Particulate matter was removed by filtration (What-man No. 1) and centrifugation (30min, 15000 xg).Supernatants were freeze-dried. Dried leachate wasplaced in sterile Petri dishes (leachate from 7.5 g perPetri dish), sterilized by 2 ml of 50% ethanol, anddried at 25''C. The Petri dish was then filled with20 ml of 0.5% malt extract agar, and inoculated withthree 5-mm agar discs overgrown with a pure fungalculture. Four species of aquatic hyphomycetes wereused, each isolated from a single conidium from theBoss Creek. Three replicate plates were used. Theplates were incubated at 20°C, and radial growthmeasured after 11 days.

For the field experiment, individual leaves wereplaced in nylon bags (10 x 10cm; mesh size 1 mm).The bags were attached to bricks and placed in theBoss Creek, a softwater stream in Fenwick, NovaScotia, Canada. Information on the biology andchemistry of this stream was given by Barlocher(1987).

After recovery of a sample (see below for schedule),five leaves of each treatment were taken from in-dividual bags, rinsed in distilled water to removedebris, placed in separate Erlenmeyer flasks (500 ml,filled with 300 ml sterile, distilled water) and aeratedfor 48h at 15°C. Each leaf was then collected, driedand weighed, and the supernatant was filtered throughan 8-[im membrane filter. Conidia of aquatic hy-phomycetes trapped on the filter were stained withacid fuchsin in lactophenol (Barlocher, 1991). Thenumber of conidia produced per mg dry weight ofleaf was determined by counting all conidia on asmall section of each of the filters. To determine therelative frequencies of occurrence and numbers ofspecies, the first 500—1000 spores encountered wereidentified.

For direct observations, three leaves of each treat-ment were rinsed and placed in separate Petri dishesfilled with sterile distilled water. After 2—4 days at15''C, the leaves were scanned for fungal structureunder a dissecting microscope. Where necessary foridentification, a small leaf section was cut out andexamined under higher magnification.

Maple leaves were placed in the stream on 4 October1990, and samples were recovered after 7, 14, 28, 56and 84 days. Stream temperature was measuredweekly at 10 a.m. The initial placement and all samplecollection dates of birch and elm leaves were delayedby 2 days.

Because exposure to frost might influence theleaching behaviour of the leaves (Barlocher, 1991),overnight air temperature was monitored with aminimum-maximum thermometer. These measure-ments were started on 1 September, 3 weeks beforethe first leaves were collected for the leaching ex-periments, and continued until 7 November.

Results

Air temperature remained above freezing until 21October, when the minimum dropped to —4°C.None of the leaves used in this study had thereforebeen frozen before collection. By 21 October, ap-proximately 50% of the maple and elm and 30% ofthe birch leaves had been shed. The temperatureagain dropped below freezing on 1 November (-5°C),2 November (-lO^C) and 7 November ( -2^) . Over90% of maple and birch leaves, and 70% of elmleaves, had fallen by 1 November, and all leaves hadbeen shed by 7 November. The daytime temperatureof the stream water declined from about 12°C to closeto zero between October and early January.

Weight losses due to leaching of the leaves aresummarized in Fig. 1. A separate one-way ANOVAwas performed for each of the three leaf species. Inall cases, there were statistically significant differ-ences among the three treatments (birch and maple:P< 0.001; elm: P<0.05).

Fig. 2 shows the number of conidia produced permg leaf material. A separate one-way ANOVA of logtransformed values was performed for each of thethree leaf spedes on each collection date. Significantdifferences (P = 0.01) in conidium productions fromfresh, dried and frozen leaves were found in all butthe fourth sample (56 day) with birch, in the first

Leaching and fungal colonization of leaves 3

30

25

20

10

Birch Elm Maple

Fig. 1. Weight lost due to leaching for 72 h at 5''C of fresh,dried and frozen leaves, n = 20, means + 95% CL. •, fresh;dried; •, frozen.

sample (7 day) with elm, and in the first and fourthsample with maple. After 84 days in the stream, elmand maple leaves were too fragile to allow aerationand subsequent filtration.

In contrast to dried and frozen leaves, fresh leavesretained some of their pigments for 1 {maple)-3(birch, elm) weeks after immersion, before turninguniformly brown.

Table 1 lists the five dominant fungal species (basedon conidium production) on the various leaf types.AnguiUospora filiformis was clearly the dominantspecies in all treatments and all three leaf species. Ittended to decline in the later stages of decay, whenArticulospora tetracladia became increasingly common.Helimcus lugdunensis, however, was more common inthe early samples, especially on birch leaves. A fourthcommon species, Flagellospora curvula did not showany consistent preference for any stage of decay.

Direct observation under the dissecting microscopegenerally revealed the same species (identificationwas based on conidiophores with attached conidia).However, the dominance of Anguiltospora filiformisand Flagelhspora ciiri'ula was less pronounced, in-dicating that these two species are very prolific sporeproducers.

Fig. 3 shows the effect of leachate from birch,maple and elm leaves on the growth of four fungalspecies. A separate one-way ANOVA was performedfor each of the four fungi. In al! cases, there werestatistically significant differences among the fourtreatments (P < 0.0001). Duncan's multiple-range

E 100

0.01

Birch

E 100

O.OI

20 40 60 80 100

Elm

20 40 60

100

0.01

80

Mople

100

20 40 60 80 100Days

Fig. 2. Mean numbers {n = 5) of conidia produced in 48h permg of leaves exposed in Boss Creek for up to 84 day, —• —,fresh; —o —, dried; —0'—, frozen.

tests indicated that with the exceptions of Articiilos-pora tetraciadia on birch and maple extracts, andAnguillospora filiformis on birch and elm extracts, the

Table 1 Percentages of conidia produced by the five dominant fungi on fresh, dried or frozen leaves of birch, elm and maple leaves

Stream exposure in weeks

BirchAnsuillospora fiUformis

a r J .'

GreatheadArticulospora tetracladia IngoldClavarioj.'sis aquatica de Wild.Clavatospora longibrachiata

(Ingold) Marvanov4 et NilssonCulicidospora aquatica PetersenFusarium sp.Flagellospora curvula IngoldFontanaspcra eccentrica

(Petersen) DykoHeiiscus lugdunensis

Sacc. & TherryLemonniera aquatica de Wild.Margaritispora aquatica IngoldMycocentrospora sp.Pyricularia aquatica IngoldTriscelopliorus sp.Number of spedes per leafTotal number of spedes on

five leavesElm

Alatospora acuminata IngoldAnguillospora filiformisArticulospora tetracladiaClavatospora longibrachiataCrucella subtilis Marvanov^

& SuberkroppFusarium sp.Flagellospora curvulaHeiiscus lugdunensisLemonniera aquaticaMargaritispora aquaticaPyricularia aquaticaTaeniospora gracilis Marvanov^Jetrachaetum elegans IngoldTripospermuni sp.

Spedes number per leafTotal spedes number on

five leavesMaple

Alatospora acuminataAiiguiltospora filiformisArticulospora tetracladiaClavariopsis aquaticaClavatospora longibrachiataDimorjjhospora fbtiicola TubakiFlagelloapora curvulaFontanasfH)ra eccentricaHetiscus lugdunensisLemonniera aquaticaMargaritispora aquaticaMycocentrospora acerina

(Hartig) DeightonPyricularia aquaticaSpedes number per leafTotal spedes number on

five leaves

Fresh

1

72 S

27.5

1.0

2

100.00.2

1

99.6

0.4

1.0

2

2

67.4

4.6

18.0

10.02.0

4

0.176,7

15,40.17,7

4.6

5

%.4

1.30.40.7

5.4

10

4

58.7

3,0

24,6

9.1

2.1

5.0

8

42.93.0

32.7

3.615,8

8.4

14

77.87.2

13.7

0.9

0.1

4.8

7

8

66.2

20.3

5.7

4.2

3.1

6.2

11

49.221.3

0.8

20.23.7

3.8

10

78.111.9

6.12.0

0.8

4.8

9

12

43.5

36.35.3

6.4

5.5

5.0

8

Dried

1

20.0

23.7

56.3

1.5

3

81.0

19.0

1,2

2

97.00.1

0.8

2.1

2.4

4

2

65 5

4.00.1

30.3

0.1

2.2

5

53,5

46,10.1

0.1

0.1

4.8

6

92.72.4

0.42.6

0.8

4,0

9

4

74.8

7.0

15.1

0.3

0.3

6.0

10

67.63.6

24.1

1.41.6

7.2

11

91.06.40.1

1.9

0.3

4.8

9

8

40.3

36.9

9.0

10,6

1.3

4.8

12

21.750.1

5.1

15.1

6.2

4.0

8

56.538.6

0.4

0.83.2

5.0

8

12

52.0

36.96.1

0.9

3.7

4.0

6

Frozen

1

51.7

6,1

42.2

2.1

3

77.7

9.8

12.5

1.4

3

99.00.1

0.9

1.4

3

2

53.5

4.0

33.19.4

2.2

4

4.443.6

40.0

9,4

1.5

3.8

7

0.1%.8

1.6

1.3

0.1

3.2

7

4

71.8

1.6

24.0

0.1

2,2

4.6

8

46,46.8

36,1

1.43.6

7.2

12

81.21.2

16.9

0.4

O.I

4.4

8

8 12

52.6 42.8

38.9 38.9

5.3 16.1

1.02.3 1.0

0.3

5.0 2.8

11 6

43.346.5

0.5

5.9

3.1

3.8

8

78,714,3

2J

1.1

1.33.6

8

i.a

1.0

0 8

0.6

g 0.4

0.2

Fig. 3. Growth rates (mm day ') of Anguillospora filijbrviis(Af.), Articulospora tetracladia (A.I.), Clavariopsis aquatica (C.a.),and Heliscus lugdunensis (H.L) on malt extract agar enrichedwith extracts of birch, elm or maple leaves, n = 9, means +95%CL. H, control;*, birch; • , elm; • , maple

growth rates of all four treatments were differentfrom each other (P = 0.05).

Discussion

Leaf-fall is preceded by senescence. This is an orderlyprocess and requires maintenance of cell functionand structure (Matile, 1986). Death of the leaf gen-erally occurs when phenolic compounds are releasedfrom vacuoles and make contact with phenoloxidases,resulting in the browning of the leaf. In many treespecies of temperate regions, this does not happenuntil some time after abscission. Drying, however,rapidly disrupts internal lamellar structures of leavesand other plant parts and increases leakage of solutesthrough the plasmalemma (Bewiey, 1979). Becausethe majority of leaves enter streams immediatelyafter detachment from riparian trees (Fisher, 1977),drying leaves, and thus accelerating damage to theirinternal structures appears inappropriate. Gessner &Schwoerbel (1990) and Gessner et al. (1991) showedthat in alder and willow leaves, the initial leachingphase is largely an artefact of this conventionalpretreatment. With the present study, this statementcan be extended to maple and birch, and to a lesserdegree, to elm leaves (Fig. 2).

Like desiccation, freezing can disrupt the internalorganization and structure of leaves and increasemembrane permeability (Burke et al., 1976). Notsurprisingly, it resulted in accelerated leaching in

leaching and fungal colonization of leaves 5

alder (Barlocher, 1991), birch, elm and maple leaves(Fig. 2). Of course, this raises the possibility that it islargely irrelevant whether or not leaves are driedbefore being placed in streams, because they mayalready have been frozen before collection. In thepresent study, approximately 50% of elm and mapleleaves, and 30% of birch leaves were shed before thetemperature dropped below 0.

In order to minimize initial variability, it is oftenstandard practice to use only unblemished leaves.As pointed out by Boulton & Boon (1991), this maygive misleading information in systems where heavyfeeding by insects or other herbivores is common.Herbivory can alter the chemistry of uneaten leavesas well as of newly produced leaves (Edwards &Wratten, 1983; Choudhury, 1988; Irons, Bryant &Oswood, 1991). This in turn can affect leaf processingin streams (e.g. Irons et al., 1991). In the presentstudy, insect damage was most visible in elm leaves.The use of naturally damaged leaves may have con-tributed to the relatively small effect that drying orfreezing had on leaching in elm leaves (Fig. 2).

Delayed leaching in fresh leaves means that solublesubstances are retained in the leaf for longer periodsof time. These substances include phenolics, aminoacids and carbohydrates (Suberkropp et ai., 1976).Some are likely to enhance the nutritional value ofthe leaf and therefore accelerate decomposition;others, especially phenolics, probably have theopposite effect. In alder leaves, the net result of theabsence of leaching in fresh leaves was delayed col-onizaton by aquatic hyphomycetes (Barlocher, 1991),which was apparently due to the prolonged presenceof antifungal substances in the leaf (Barlocher, 1990).The same effect was observed with birch, elm andmaple leaves (Fig. 2, Table 1). After 7 days in thestream, conidium production from dried leaves ex-ceeded that from fresh leaves by a factor between6 (maple) and 2020 (elm). The difference betweenfrozen and fresh leaves was not quite as pronounced(3.4 on maple to 420 on elm). With birch, these dif-ferences persisted for at least 4 weeks; in maple andelm leaves they had disappeared by the second week.In the later stages of decay (54 days with maple,84 days with birch) more conidia were producedfrom fresh leaves. These results indicate that fungalcolonization will be overestimated during the earlystages, and underestimated during the later stages,of decay when dried instead of fresh leaves are used.

6 F. Barlocher

Delayed fungal colonization will likely result in de-layed invertebrate feeding. Decomposition ratesaveraged over the entire period may well remainunchanged; nevertheless, in temperate climates moreof the actual decomposition will shift toward thecolder months. In addition, the quantity of solubleorganic matter released from leaves and potentiallyavailable to epitlithic communities will be reduced.

Aqueous leaf extracts inhibited growth of fourcommon aquatic hyphomycetes (Fig. 3). The weakesteffect was found with elm leaves, and the strongesteffect with maple leaves. Nevertheless, fungal col-onization of fresh leaves was most delayed in birchleaves. This suggests that in addition to the potencyof the extract, their retention by the leaf is important.As mentioned earlier, fresh birch and elm leavesretained their integrity (judged by presence of pig-ments) for up to 3 weeks, and maple leaves for only1 week.

Anguillospora filiformis was one of the dominantspecies in most samples (Table 1). It tended to declinein the later stages of decay; not surprisingly, thissequence was somewhat delayed in fresh comparedto dried and frozen leaves. Heiiscus lugdunensis wasless dominant, and its drop in later samples morepronounced. By contrast, Articulospora tetracladiabecame more common in the later samples. Thesethree spedes had shown similar patterns on alderleaves in a Dartmoor stream (Barlocher, 1991). In thatstudy, however, Lunuhspora cunmla had also beenone of the dominant species. It was completely absentin Boss Creek, probably because it requires a rela-tively high temperature for growth and reproduction(Suberkropp, 1984; Webster, Moran & Davey, 1976).

Of special interest was the appearance of the fungusCrucella subtilis. This species has only recently beendescribed from various streams in Alabama (Marva-nov^ & Suberkropp, 1990). It appears to be an un-specialized parasite of other aquatic hyphomycetes.

Results in fungal ecology often depend strongly onthe technique used. Traditionally, the accepted (ifseldom attained) goal has been to base dominancepatterns on biomass present in the form of metab-olically active mycelium. Here, it was based on anestimate of reproductive potential. I believe thisis justified, because there is little delay betweengrowth and reproduction in aquatic hyphomycetes(Suberkropp, 1991). In addition, it appears obviousthat for aquatic hyphomycetes spore production is

essential for dispersal and ultimately survival in astream. Nevertheless, dominance patterns estimatedin this manner remain relative, because it is im-possible to reproduce conditions prevailing in thestream (e.g. microcurrents, water chemistry, etc.) thatare known selectively to inhibit or stimulate repro-duction of different species. Higher spore productionin itself does not automatically increase the fitnessof a fungus; other factors involved include sporeviability and their ability to settle and germinate onappropriate substrata.

Overall, the results confirm that decreased leachingin fresh leaves means that, during the first few daysto weeks, fewer aquatic hyphomycete species will beable to colonize, and that those present will producefewer spores. It is possible that other microbialgroups (e.g., bacteria, Oomycetes, yeasts) will profitfrom the retention of soluble substances.

Even though most leaves enter a stream duringautumn, storms can introduce green leaves through-out the year. The changing chemical composition ofleaves during a growing season, partly in responseto herbivore attack (Edward & Wratten, 1983; Choud-hury, 1988; Irons ct al., 1991) may well change theconsequences of drying leaves for fungal coloniz-ation. This aspect is being investigated.

Acknowledgments

The financial support of the Natural Sciences andEngineering Research Council of Canada is gratefullyacknowledged.

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(Manuscript accepted 12 January 1992)