Leaf Barriers to Fungal Colonization and Shredders (Tipulalateralis) Consumption of Decomposing Eucalyptus globulus

C. Canhoto, M.A.S. Graca

Department of Zoology, University of Coimbra, 3000 Coimbra, Portugal

Received: 8 July 1998; Accepted: 21 December 1998

A B S T R A C T

Herein we assess the importance of leaf cuticle, polyphenolic, and essential oils contents of Euca-

lyptus globulus leaves to hyphomycete colonization and shredder consumption. Optical and electron

microscopy revealed that, at least during the first 5 weeks of conditioning, the cuticle remains

virtually intact. Stomata provide the main access for hyphae to internal leaf tissues and, eventually,

for spore release. We suggest that in E. globulus leaves, fungal decomposition progresses predomi-

nantly in and from the eucalyptus leaf mesophyll to the outside. Malt extract agar media supple-

mented with either eucalyptus essential oils or tannic acid completely inhibited (Articulospora

tetracladia, Lemonniera aquatica, and Tricladium gracile) or depressed (Heliscus lugdunensis, Lu-

nulospora curvula, and Tricladium angulatum) aquatic hyphomycetes growth. The transference of

both secondary compounds to alder leaves induced similar and significant reduction in Tipula

lateralis larval consumption. Results consistently indicate that eucalyptus oils are stronger deterrents

than polyphenols. The waxy cuticle of E. globulus appears to be a key physical factor delaying fungal

colonization during decomposition. We hypothesize that the relative influence of leaf phenols and

essential oils to aquatic hyphomycetes and shredders may be related to three main factors: (a) initial

distribution of such compounds in the leaves; (b) possibility of their decrease through decompo-

sition; and (c) consumption strategies of detritivores.

Introduction

Decomposition of Eucalyptus globulus Labill. leaves have re-

cently been investigated in Brazil [28], Spain [4], India [36],

and Portugal [3, 14] where large areas of the native flora

have been converted to eucalyptus plantations. Being the

dominant plant material in most riparian areas of Central

Portugal, originally shaded by deciduous forests, eucalyptus

detritus may represent the major energy source for stream

communities [1].

Several comparative studies have classified the leaves of E.

globulus in the medium or slow processing category [11, 14,

25, 36] and have shown that particular chemical and physical

leaf features may influence decomposition [3, 11, 18, 35].

Although high numbers of fungal conidia can be producedCorrespondence to: C. Canhoto; E-mail: cmcanhoto@gemini.ci.uc.pt

MICROBIALECOLOGY

Microb Ecol (1999) 37:163–172

DOI: 10.1007/s002489900140

© 1999 Springer-Verlag New York Inc.

on decomposing E. globulus, the leaves have been found to

delay aquatic hyphomycete sporulation [3, 14, 36] and re-

strict shredder consumption [12, 13]. Relatively high levels

of tannins and essential oils accumulated in leaf glands,

along with a thick cuticle (with cutin, waxes, phenolics, pec-

tin and cellulose; [10]), limit microbial and macroinverte-

brate access to the inner tissues. However, it has been pre-

viously suggested that different feeding strategies may be

used by both consuming groups to minimize the effects of

the leaf secondary defensive compounds [3].

Herein we evaluate the importance of the eucalyptus leaf

cuticle, essential oils, and polyphenolics as barriers to fungal

colonization and shredder consumption. For this purpose,

we examined fungal colonization and eucalyptus cuticle

changes over an immersion period of 5 weeks. The ability to

grow on substrates with increasing polyphenolic and oil con-

tents was also tested with the aquatic hyphomycetes Articu-

lospora tetracladia Ingold, Heliscus lugdunensis Sacc. et

Therry, Lemonniera aquatica De Wild, Lunulospora curvula

Ingold, Tricladium angulatum Ingold, and Tricladium gracile

Ingold. To evaluate the effects of secondary compounds on

shredder consumption, we used a cranefly larva that occurs

in low order streams of Central Portugal, Tipula lateralis

Meig.

Materials and MethodsGeneral

Leaves of eucalyptus and alder (Alnus glutinosa L.) were collected

before abscission in October, air dried in the dark, and stored until

needed. For conditioning purposes, leaves were assembled in

groups of approximately 2 g (two to four leaves) in individual

nylon bags (10 cm × 14 cm; 0.5 mm mesh size). Six groups of four

bags were tied to nylon ropes and submerged in “Ribeira do Sobral

Cid” (40°68N 8°148E), Coimbra, a second-order stream of Central

Portugal. Leaf groups were immersed over time—35, 21, 14, 7, 2,

and 0 days, respectively. The six ropes were harvested at day 0 and

brought to the laboratory. Leaves were then gently washed to re-

move attached sediment. Eucalyptus leaf remains were immediately

prepared for light and electron microscopy, as indicated below.

Microscopy

Small leaf sections (1–2 mm length), obtained from at least four

different leaves (one of each bag), were cut parallel to the main

vein. The rectangles were fixed in 2.5% glutaraldehyde, post-fixed

in 1% buffered osmium tetroxide, and dehydrated in graded etha-

nol series (20–100% v/v). Additionally, and in order to observe

polyphenols in the plant tissue, nonexposed material was fixed in a

mixture of 2.5% of glutaraldehyde, 0.5% caffeine, and 1% K2Cr2O7

[46]. For SEM examinations, the leaf sections were critical-point

dried with carbon dioxide as transition fluid, coated with gold, and

mounted on aluminum stubs.

For optical and TEM microscopy, the samples, after dehydra-

tion, were embedded in Spurr’s resin [41]. For general observa-

tions, semithin sections were stained with 0.2% toluidine blue.

Cuticle sections (1–3 µm) of eucalyptus leaves were also stained

with 0.01% auramine O [29] or 0.01% benzo[a]pyrene–caffeine

and observed under an epifluorescence microscope. Ultrathin sec-

tions, cut on a ultramicrotome and collected on uncoated copper

grids, were stained with uranyl acetate and lead citrate [37]. Ob-

servations were performed in a Siemens Elmiskop-101 transmission

microscope.

Fungal Growth

To test the effects of eucalyptus leaf secondary compounds on

fungal growth, increasing concentrations (0–7.5%) of tannic acid

and essential oils were added to a malt extract agar medium (36.6

g L−1) prepared in PIPES buffer. The pH was previously adjusted to

7, with NaOH, and the medium sterilized, at 120°C, for 15 min.

Essential oils were extracted by steam distillation from leaves that

had been air dried for 3 days [25].

Laboratory cultures of Articulospora tetracladia, Heliscus lug-

dunensis, Lemonniera aquatica, Lunulospora curvula, Tricladium an-

gulatum, and Tricladium gracile were grown in malt extract agar

media, at 20°C. Small pieces, cut from the edges of the colony, were

used as inoculum in the center of the agar plate (at least 3 repli-

cates/treatment). Fungal growth was allowed for 15 days and ex-

pressed as the increase in the colony size (squared mean diameter,

cm2).

For each species, the significance of the effect of tannic acid or

oils on fungal growth was determined using one-way ANOVA (log

(x+1) transformed data) followed by a Student–Newman–Keuls test

[45]. The EC50 values (from the relationship probit transformation

of effect percentage vs log concentration of secondary compound)

were obtained using a probit analysis [23].

Consumption Experiments

The effect of E. globulus polyphenolics and essential oils on feeding

rates of T. lateralis larvae was studied. Larval specimens of this

family present a high midgut pH that makes them potentially well

adapted for the digestion of leaf proteins usually found complexed

with lignins and polyphenols (e.g., [31]).

Larvae were collected, in autumn, from “Ribeira de S. Joao”

(40°118N 8°258E), Lousa, a low-order stream in Central Portugal.

They were acclimatized to laboratory conditions (15°C; 12:12 h

light/dark photoperiod) for 1 week. Specimens were then allocated

individually to 70 cm diameter × 85 cm high plastic containers

filled with 250 ml of aerated artificial pond water (APW: Ca, 80 mg

L−1; Cl, 145 mg L−1; Mg, 12 mg L−1; Na, 18 mg L−1; K, 3 mg L−1;

pH 7.9). The bottom of the containers was covered with a layer of

164 C. Canhoto, M.A.S. Graca

fine, ashed stream sand (500°C; 8 h). Larvae were starved for 48 h

prior to the experiments.

Food was supplied as leaf disks (2 per larva) cut from leaves

conditioned (i.e., colonized by microorganisms) in the same stream

for 3 weeks, dried, and weighed to the nearest 0.01 mg. Controls

consisted of leaf material without larvae. After 3 days, the remain-

ing leaf material was retrieved, dried, and weighed. Individual con-

sumption (mg) was estimated as the difference between the initial

and final dry weight of the leaves, corrected from controls, and

expressed per mg dry weight on individuals, per day.

One-way ANOVA (log (x + 1) transformed data), followed by

Tukey’s test [45], was used to compare feeding rates.

Polyphenolics

Polyphenolic solutions were obtained from unconditioned euca-

lyptus leaves (≈40 g). Extraction was performed twice in 50% ac-

etone at 70°C for 20 min. The combined volumes of the solvent

were allowed to evaporate for 1 week. The compounds that re-

mained adherent to the flask walls were subsequently dissolved in

200 ml of 50% acetone. Total phenolics of the stock solution (16.2

mg ml−1) was estimated using the Folin Denis reagent [40]. Tannic

acid was used as standard.

According to previous work (e.g., [4]), eucalyptus polyphenolics

usually represent about 10% of leaf dry weight. Keeping this value

as a reference, we used the stock solution to increase alder phenolic

values to 5% (A5%), 10% (A10%) and 25% (A25%) of leaf dry

weight. The eventual influence of the solvent on shredders con-

sumption was tested with alder disks soaked with identical increas-

ing volumes of 50% acetone (10 replicates of each). Acetone was

always allowed to evaporate before consumption.

Essential Oils

The influence of essential oils on shredder consumption was as-

sessed with a total of 140 larvae that were individually fed alder (A),

alder impregnated with eucalyptus essential oils (A + O), eucalyp-

tus (E), or eucalyptus without oils (E − O).

Essential oils can reach 5% of eucalyptus adult leaf mass; how-

ever, intrinsic (e.g., leaf age) or extrinsic (e.g., temperature) factors

may change leaf oil content [19]. In our case, we obtained 2 ml of

essential oils per 100 mg of eucalyptus adult leaves. Thus, the oil

content of a mean eucalyptus circle (18.21 mg ± 0.6 SE; n = 50) was

0.364 µl.

Alder disks were treated with 0.8 µl of 1:1 essential oil/ether

solution. Ether was allowed to evaporate for 10 s and the two

moistened disks immediately immersed for consumption. As con-

trols, alder leaves treated with 0.8 µl of 50% ether were offered as

food to T. lateralis larvae (n = 10).

Results

Transverse sections of eucalyptus leaves showed a continu-

ous thick cuticular membrane over the leaf epidermis with

thinner extensions lining the substomatal cavities or between

cells (Fig. 1a). This waxy cuticle layer was resistant to high

water temperatures (used in the steam distillation process)

and remained practically unaltered through 5 weeks of im-

mersion. At this stage, leaf mesophyll was clearly degraded

and missing. Under manipulation, the leaf vascular system

was easily detached from the cuticles of both leaf surfaces,

making observations very difficult.

Contrasting with the restricted presence of essential oils

in vesicles (Fig. 1b), leaf polyphenols appear to be spread in

the leaf mesophyll cells, mainly adjacent to the cell walls and

in vacuoles (Fig. 1c).

Colonization was apparent soon after immersion. Hy-

phae were detected on the leaf cuticle, beneath the cuticle,

between the epidermal cells, and inside the plasmolized leaf

tissues, after 2 days. Stoma and, later on, fissures on the leaf

surface, were the only evident avenues for mycelial penetra-

tion (Fig. 2a–d). Invasive fungal hyphae showed digestion

activity at least after 2 weeks of immersion (Fig. 3a). TEM

observations of electron-dense sheathing material was fre-

quently detected around the hyphae and adjacent to the

degraded cell walls and middle lamella (Figs. 3b, c).

Eucalyptus secondary compounds completely inhibited

or depressed fungal growth (Figs. 4a, b, c). Articulospora

tetracladia, L. aquatica, and T. gracile did not grow in media

supplied with tannic acid or oils. Heliscus lugdunensis, L.

curvula, and T. angulatum growth were significantly de-

creased by the addition of either oils or tannic acid (P <

0.001). Higher percentages of tannic acid in relation to es-

sential oils were always needed to inhibit fungal growth—

0.75% for L. curvula and 0.25% for T. angulatum and H.

lugdunensis (vs 0.1% oils in all cases). In fact, low and similar

values for EC50 were obtained when oils were present: 0.248

(0.086–0.717, 95% CL) for H. lugdunensis, 0.290 (0.186–

0.45, 95% CL) for L. curvula and 0.358 (0.234–0.55, 95%

CL) for T. angulatum. Lunulospora curvula was the most

tolerant species to tannic acid (EC50 = 2.202; 1.56–3.104,

95% CL) followed by H. lugdunensis (EC50 = 1.1914; 0.83–

4.43, 95% CL) and T. angulatum (EC50 = 0.528; 0.39–0.715,

95% CL).

The addition of polyphenols to alder significantly de-

creased Tipula consumption (P < 0.001; Fig. 5). When poly-

phenol content reached values similar to those usually found

in unconditioned eucalyptus leaves (≈10%), consumption

declined to 50%. Increased oil content in alder leaves also

depressed larva consumption (P < 0.001; Fig. 6). A 50%

reduction in consumption was obtained when specimens fed

Leaf Barriers to Eucalyptus Decomposition 165

alder discs treated with essential oils (A + O). On the other

hand, consumption of leaves increased when essential oils

were previously extracted (E − O).

The deterrent role of oils was corroborated by occasional

observations of chironomids feeding on decomposing E.

globulus leaves. Because of their small size, these inverte-

brates are able to consume these leaves discriminately,

avoiding the oil glands (Fig. 7a, b).

Fig. 1. Transverse sections of dried unconditioned

Eucalyptus globulus leaves. (a) The leaf was stained

with benzo[a]pyrene–caffeine (×214). The cuticle

(C) can be seen as a white fluorescent layer covering

the epidermal cells (EC). Thinner extensions (arrow)

of the cuticle occur lining stomata (S) and coating

the walls of the substomatal chambers (SC). (b) A

large oil vesicle (OV) cavity can be observed in this

section stained with auramine O (×107). (c) Leaf

section fixed in glutaraldehyde, caffeine, and

K2Cr2O7 and stained with toluidine blue (×428).

Darker areas (arrows) seem to indicate phenolic pre-

cipitation formations. (All magnifications original.)

166 C. Canhoto, M.A.S. Graca

Discussion

Waxes, cutin, polyphenol compounds, and oils are usually

considered as inhibitors [32, 39] of leaf conditioning in

streams (process of microbial colonization and growth in

detritus; [6]). Eucalyptus colonization seems primarily con-

trolled by the presence of a resistant waxy cuticular mem-

brane that hardens eucalyptus leaves [26] and reduces hy-

phal penetrating capacity.

According to our observations, and in agreement with

Gallardo and Merino [24], fungal invasion appears to be,

most of all, a mechanical process that takes place, after im-

mersion, through stomata and, when present, through fis-

sures on the leaf surface. Although not evident, the addi-

tional involvement of fungal enzymes in cutin degradation

must not be excluded [7, 9]; some species of fungi are known

to produce cutinases [27]. Stomata, natural discontinuities

on eucalyptus leaves, may facilitate conidia settling and ger-

mination and provide an easier route of entry (and, even-

tually, a way out) for hyphae. In terrestrial ecosystems, im-

permeability of waxes is known to reduce leaf infections by

preventing deposition of water-borne inocula [30]. A con-

strained (stomatal) area of fungal access to the leaf meso-

phyll is, most probably, the primary course of the delayed

conidia production usually observed in decomposing euca-

lyptus leaves [3].

Fig. 2. Eucalyptus leaves after 2 weeks of immersion. (a) Transverse section stained with toluidine blue. Hyphae (HY) can be observed over

the cuticle, in the damaged mesophyll and mycelium passing through a stoma (×107). (b) Higher magnification hyphae (HY) passing

through a stoma (×1070). (c) Scanning electron micrograph of hyphae (HY) crossing a stoma (S) (×2250); (d) Scanning micrograph of a

section of a detached epidermis (internal view) from the abaxial leaf surface with hyphae (HY) passing through stomata (S) to the leaf

mesophyll (×1125). (All magnifications original.)

Leaf Barriers to Eucalyptus Decomposition 167

Contrary to early claims (see [8]), there is no doubt that

aquatic hyphomycetes play a major role in the decomposi-

tion of eucalyptus leaves, at least in Portuguese streams. As

degradation proceeds, the fungal attack (presumably facili-

tated by a cracking persistent waxy cuticle along with a re-

duced polyphenolic content) gradually disrupts and digests

the inner leaf tissues, making leaves softer. Toughness is

usually negatively related with fungal colonization and in-

vertebrate consumption [20, 21, 33]. However, the contri-

bution of fungal enzymes to leaf maceration has been dem-

onstrated [15, 17, 26, 38, 43]. The role of bacteria in this

process should not, however, be neglected; enzymes such as

pectinases or polygalacturonate lyases may be common in

this group (e.g., [16]).

The role of polyphenols and terpenes as defensive com-

pounds against fungi is generally accepted [3, 5, 8] and was

corroborated by our study. The addition of oils or tannic

acid to the malt extract agar totally inhibited growth of three

species and decreased mycelial growth of H. lugdunensis, L.

curvula, and T. angulatum. In these last cases, however, the

eucalyptus essential oils (mainly constituted by cineole and

pinene; [19]) showed a stronger depressing effect on fungal

growth than polyphenols. Indeed, in terrestrial and marine

environments terpenes are also often more deterrents than

phenols [42]. Fungal sensitivity to phenols seems to be spe-

cies specific. Heliscus lugdunensis (an earlier wood colonizer)

was, in fact, previously referred as a tolerant species [2]. The

ability to tolerate leaf defenses (possibly related with differ-

Fig. 3. Hyphal digestive activity in eucalyptus leaves immersed for 2 weeks. (a) Transverse section (stained with toluidine blue) of fungal

hyphae (HY) showing the digestion of eucalyptus parenchyma cells (PC). Digested areas are indicated by arrows (×1070). (b) Transmission

electron micrographs of transverse sections of hyphae (HY) digesting microscopy cell wall (CW) (×25,200) and (c) the middle lamella (ML)

(×20,000). Note the cavity formed between the cell walls, probably by hyphal digestion, and the middle lamella granular wall lysate. The

hyphal sheath (HS) is clear in both micrographs. (All magnifications original.)

168 C. Canhoto, M.A.S. Graca

ential enzymatic abilities) may allow an earlier colonization,

which may constitute a competitive advantage in eucalyptus

conditioning [3]. In fact, in a earlier study, Canhoto and

Graca [14] showed that sporulation of eucalyptus leaves was

retarded by 2 weeks when compared with alder leaves. In the

same study, Heliscus lugdunensis was the first species colo-

nizing eucalyptus leaf packs.

A similar qualitative effect was produced by both euca-

lyptus secondary compounds on Tipula consumption. How-

ever, it is interesting that small increases in alder phenolic

content (ø5% leaf dry weight) do not significantly affect

shredder consumption: the leaves’ high nutritional value

may compensate the deleterious effect of phenols. Indeed,

phenol deterrency is expected to be more effective in sub-

strata of poor quality [34]. We hypothesize that shredder

digestive physiology (e.g., a high midgut pH) and leaf nu-

tritional value may be important factors determining the

level of phenolic tolerance for each species. The real ecologi-

cal importance of oils as feeding deterrents in a natural lotic

environment may also be ruled by larval feeding strategies.

According to our observations, T. lateralis shred the leaf with

aleatory bites; the ability of larvae to recognize and avoid

such confined compounds is not evident. In contrast, a clear

avoiding behavior toward oil vesicles was adopted by Chi-

ronomidae-fed eucalyptus (see also [11]).

In summary, we propose that E. globulus breakdown is

mainly ruled by a preinfectional barrier constituted by a

resistant cuticular layer that effectively isolated the leaf ma-

trix. The resultant elongated physical (and, possibly, chemi-

cal) integrity may retain eucalyptus leaf defenses, retarding

microbial attack, shredder consumption, and biological frag-

mentation. Stomata seem to be the major route for fungal

Fig. 4. Aquatic hyphomycete growth (cm2) in malt

extract agar medium supplied with increasing con-

centrations (0–7.5%) of tannic acid (white bars) and

eucalyptus essential oils (black bars). Growth was al-

lowed for 15 days. (a) Heliscus lugdunensis. (b) Lu-

nuslospora curvula. (c) Tricladium angulatum. Values

are means ± SE. The symbols * and # indicate the

lowest concentration of tannic acid and oils, respec-

tively, that induced a significant (P < 0.05) decrease

in fungal growth.

Leaf Barriers to Eucalyptus Decomposition 169

access; however, their role in conidia release to the exterior

is also conceivable and needs further attention.

The hypothesis that fungal decomposition of eucalyptus

leaves proceeds from the inside, primarily because of a re-

sistant cuticle, may explain the predominance of apparently

intact but “hollow” leaves that are common in Portuguese

streams. Such a breakdown process may have particular im-

portance in systems when the activity of shredders is scarce

because of the low nutritional value of eucalyptus [12,13] or

the low densities of this feeding group [44].

Acknowledgments

We thank Dr. Felix Barlocher for valuable comments on the

manuscript. We also thank Mr. Jose Dias who provided

technical assistance and Professor Jose Mesquita for the

comments and use of laboratory facilities. This work was

supported by JNICT (project number PBIC/C/BIA/2056/95)

and IMAR.

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