Introduction

Bark presents a formidable barrier providing both constitutive and induced defence mechanisms. Despite, or perhaps because of, its ubiquity and effectiveness in protecting the underlying tissue, there have been, according to Pearce (1996), a few detailed studies of the constitutive protection provided by secondary plant surfaces (cf. Campbell et al. 1980; Pearce 1987; Merrill 1992). Few fungi appear to be able to penetrate an intact bark surface directly from spore inoculum (Dickinson 1976); one example is Heterobasidiom annosum s.l. (Peek et al. 1972).

Several studies have already been performed on bark extracts of different tree species. A majority of the studies deal with tropical tree species, and studies of antimicrobial activity against bacteria and fungi related to humans are most frequent. (Rovira et al. 1999) tested the antimicrobial activity of 203 neotropical wood and bark extracts against a panel of four human pathogens.

Some studies have looked into the effect of bark extracts on plant pathogenic and wood decaying fungi. Kofujita et al. (2002, 2006) found moderate antifungal activities of a diterpene quinone from the bark of Cryptomeria japonica D. Don against plant pathogenic fungi. In Kofujita et al. (2001), the fungal growth was inhibited by addition of C. japonica bark meal. Different extraction solutions were tested and the strongest activity was found from hexane extract. An antifungal compound isolated from the hexane extract was identified as ferruginol, which was assumed to play an important role in the antifungal activity of the bark. Kokalis-Burelle and Rodriguez-Kabana (1994) evaluated the effects of pine bark extracts and pine bark powder from Pinus taeda L. and Pinus elliottii Engelm. The growth of all the tested fungal plant pathogens was inhibited by pine bark powder amended media. Fungal pathogens differed in their response to compounds extracted from bark. Mori et al. (1995, 1997) screened acetone extracts from the barks of 51 different deciduous and 21 conifer tree species on seven plant pathogenic fungi and four wood decay fungi. In general, deciduous trees showed only weak activity, except for Magnolia. The extracts of the Pinaceae family showed some activity against each of the tested fungi regardless of the genera or species of the trees. However, the extracts inhibited the growth of plant pathogenic fungi more effectively than that of wood decay fungi. The antifungal activity of the Pinaceae was superior to that of the Cupressaceae, which according to Mori et al. (1995) is generally known to have potential antifungal activity.

Few studies have been published on the antifungal effect of bark extracts of the natural inhabiting wood species of northern Europe. The coniferous Larix decidua Mill. and Picea abies (L.) H. Karst. grown in Japan have already been tested by Mori et al. (1995). The aim of this study was to screen the antifungal effect of bark extracts from some European tree species. The effect of the bark extracts was tested against fungi in different damage categories: brown rot, white rot, canker and blue stain. The results are discussed with focus on the bark extracts effect on different damage categories, the effect of the main host and also in comparison with the results of Mori et al. (1995, 1997).

Material and methods

Bark extracts

All the plant materials (Table 1) were collected at Rogaland Arboretum, Sandnes, in the western part of Norway during November and December in 2000. The age of the test trees was approximately 10 years. All the bark samples were collected from the stems of the trees. All the tested tree species are native Norwegian species except for Larix decidua. Parts of the stems were debarked, and the bark samples (2.0 kg each) were splintered by the use of an electric garden splinter machine. Each sample was extracted twice in 5.0 l of dichloromethane (PA quality, Prolabo, France), then twice with 5.0 l of technical grade methanol (Baker, The Netherlands). The extractions occurred at 20°C for 24 h. The combined extracts were concentrated on a 20 l rotational evaporator (R220, Büchi, Switzerland) followed by concentration on a smaller rotavapor (R124, Büchi, Switzerland). The concentrated samples were dried to completeness on a Savant SpeedVac AES 2010-system (GMI, MN, USA).

Table 1 Tree species used in the experiments
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In pre-trials, bark extracts from all tree species were tested in 25, 50 and 100 mg/ml concentrations against Heterobasidion annosum and H. parviporum. Ethanol was used as solvent and control. This pre-trial study formed the basis of the bachelor’s degree work of Keuning (2001), performed at the Norwegian Forest Research Institute’s (now Norwegian Forest and Landscape Institute) laboratories in 2001. All samples were stored in a freezer until further use. In the study presented in this paper, all the fungi were tested again against 100 mg/ml and 25 mg/ml concentrations of the nine bark extracts. Comparison of fungal growth rates revealed that the storage of the bark extracts did not influence the activity of the samples.

Fungal strains

The fungi used in bioassaying the bark extracts are listed in Table 2. Fungi from different damage categories (brown rot, white rot, canker and blue stain) were selected. All strains are isolated, identified and stored in the Norwegian Forest and Landscape Institute’s culture collection.

Table 2 Fungi used in the screening of bark extracts are given together with damage category, host tree preference and culture strain number in the culture collection at the Norwegian Forest and Landscape Institute
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Agar plate test

To sterile filter paper (Whatman), 1 ml of sample and ethanol solution was added. Ethanol was used as control. The filter papers were dried and then covered with a thin layer (8 ml) of 4% (w/v) malt extract-agar medium. The plates were inoculated at the centre and incubated in a dark environment at 21°C. Four replicate dishes were used. Radial growth was measured in four directions for each plate at 24-h intervals. Mean values from the last measurement (when the control reached the edge) were used in the further analysis. Antifungal activity (AFA) was calculated: AFA = 100 × (GC − GT)/GC, where GC = hyphal growth on the control medium with solvent control and GT = hyphal growth on the test medium. The mean growth rates using 100 mg/ml concentration were compared with the control in a Tukey–Kramer test.

Results and discussion

To test the reproducibility of the screening test and test solutions, growth rate results from H. annosum and H. parviporum in this study were compared with results from the same bark extracts used in a pre-trial study by Keuning (2001) (data not shown). The trend was similar in both the tests, proving the reproducibility of the test and test solutions. There was, as expected, a dose-response effect. Higher bark extract concentrations produced higher antifungal activities (AFA). The results from the highest concentration, 100 mg/ml, will be used for further discussion.

Given that the cutinized epidermis or suberized periderms of trees are the first tissues that potential pathogens encounter (Kolattukudy and Koller 1983), and given that the majority of trees remain alive for decades or centuries, these barriers are apparently very effective (Pearce 1996). The outer layers of the bark represent a constitutive defence including antifungal compounds, but the AFA of several of the north European tree species is previously unknown. The AFA of the bark extracts screened in this study are presented in Fig. 1 and the statistical comparison in Table 3.

Fig. 1
figure 1

The antifungal activity (AFA) is given in percent when exposed to the bark extractives. Results with the 25 mg/ml concentration are given by the grey bars and those with the 100 mg/ml concentration are given by the black bars

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Table 3 Tukey–Kramer comparison of the growth rate mean of the fungi with that of the control using media with nine different bark extracts
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General trends

Generally the decay fungi were more inhibited by the bark extracts than the blue-stain fungi, while the lowest inhibition were found among the cancer fungi. The route of ingress of many wood-infecting fungi remains uncertain, although entry through wounds that expose the xylem, either above or below ground, seems likely (Pearce 1996). The main pattern found between the fungal groups in this study is believed to be caused by the route of ingress. The decay fungi in this study primarily attack through wounds and roots (Woodward et al. 1998). Both blue stain and canker fungi are known to use insect vectors or wounds to access the wood (Solheim 1986; Furniss et al. 1990; Aukemaa et al. 2005). The cancer fungi often use wounds as an access pathway, but they kill the bark around the wound (Roll-Hansen and Roll-Hansen 1980; Outila 1990; Butin 1995). Hence, it is important that the cancer fungi are able to cope with the antifungal components in the bark.

The deciduous bark extracts in our test were found to be less active against the canker and blue stain ascomycete fungi than the conifer bark extracts. The exceptions in the present study were Acer platanoides in general and the high effect of Alnus incana extracts against the blue stain fungus Ceratocystis polonica. The trend found in this study between deciduous trees and conifers is partly supported by Mori et al. (1997). They found that the deciduous bark extracts showed generally less activity against both plant pathogenic and wood decaying test fungi compared to those of conifers. Mori et al. (1995) found that the extracts of the coniferous trees inhibited the growth of plant pathogenic fungi more effectively than that of wood decay fungi. This effect was not fully confirmed in this study of European species. The comparisons between our study and the Japanese studies in this paper must be seen as indicative, as the test solution preparation and test procedure differ. Other parameters that might influence the data as a result of the sampling are the age of the trees, the thickness of the bark and also the growth and the climatic conditions.

The canker and blue stain ascomycete fungi, with the exception of Nectria ditissima, have conifers as main host. Since conifers were generally more effective against these fungal groups than the deciduous trees, it might indicate a host effect, causing higher AFA from the host. However, N. ditissima has deciduous trees as host, but the lowest effect of A. platanoides is found for this species.

Bark extracts

Acer platanoides extract was clearly the most efficient of the screened bark extracts in reducing the growth of the test fungi. In Mori et al. (1997), bark extract from Acer mono Maxim. showed only minimal effect (0–25%) on their fungi. A chemical analysis of the bark extracts would most likely have given some clues about the compounds causing this high AFA of A. platanoides. No chemical analysis has been performed in this study and therefore some hypothesis is put forward based on ecological circumstances. One hypothesis is based on the nature of the bark. Base-rich deciduous tree species have significantly higher pH and availability of nutrients (e.g. nitrogen) than other tree species. These have been suggested to be the most important factors affecting species composition and diversity of lichens and bryophytes (Hallinbäck 1996). Acer platanoides host a rich epiphytic lichen (Ekman 1997) and bryophyte (Hallinbäck 1996) flora. The rich epiphyte flora on A. platanoides and availability of nutrients might be a reason for the high AFA. This might increase the risk for fungal attack and again increase the need for high AFA. The pH was measured in all the tested bark extracts and was close to pH 5 for all species (except for Betula pubescens pH 5.6 and L. decidua pH 4.3). Hence, the pH of A. platanoides bark extract alone cannot explain the high AFA in this test. Another hypothesis is with regard to main hosts. The fungi in this test have conifers as the main host except for N. ditissima. Nectria ditissima is the fungus where A. platanoides bark extracts has the lowest effect. But the assumption that the main host cause lower AFA than the other tree species is not valid for most of the other tree–fungus comparisons in this test.

In our study, B. pubescens extract mainly gave the weakest reduction in growth rate, generally less than 25% AFA, a result supported by the results of Betula species (B. platyphylla var. japonica Hara, B. davurica Pall. and B. ermanii Cham.) in Mori et al. (1997). The consequence of the weak AFA of B. pubecesns would either imply that it is easily attacked by fungi or that the antifungal components in the bark only contribute to a small part of the trees constitutive defence. The latter is the most plausible explanation; fungal attack is mainly through wounds or roots.

Quercus petraea extract produced over 25% AFA for the decay fungi and less than 25% for the other test fungi. According to Mori et al. (1997), the Quercus species (Q mongolica var. grosseserrata Rehd. & Wils. and Q. dentate Tunb.) generally had no or less than 25% effect. Quercus petraea wood is regarded as a naturally durable species; the other tested deciduous species are considered not durable (EN 350-2 1994). Quercus petraea gave slightly higher growth rate reduction among the decay fungi than the conifers, except for the effect of P. abies on Fomitipsis pinicola. For the other fungi it was opposite, Q. petrea had a lower growth rate reduction than the conifers used.

Interestingly, A. incana extract produced a 95% reduction of the growth rate of C. polonica. Alnus incana gave less than 25% AFA for the rest of the fungi except for Nectria fuckeliana with 31%, H. parviporum with 29% and H. annosum with 36%. Alnus hirsuta Turcz., in Mori et al. (1997), generally produced less than 25% AFA.

The effect of Salix caprea extract was quite weak in our study, generally less than 25% AFA, a result supported by the results of Salix species (S. pet-susu Kimura, S. hultenii var. angustifolia Kimura and S. miyabeana Seemen) in Mori et al. (1997). The exception in our study was Salix caprea extract that reduced the growth rate of C. polonica, H. annosum and H. parviporum by 40%.

There are no published reports on antifungal activity of Pinus sylvestris extracts, while Japanese samples of P. abies and L. decidua were screened by Mori et al. (1995). The AFA results of P. abies and L. decidua in our study were quite similar to the results of Mori et al. The three tested decay fungi have conifers as main hosts. However, conifer extracts had a quite similar effect to most of the deciduous tree species extracts against the basidiomycetes.

Decay fungi

For H. annosum, no significant difference was found between the three coniferous species. Heterobasidion annosum is known to have a broader host range than H. parviporum (Korhonen et al. 1998). The main host of H. parviporum is P. abies, occasionally also infecting P. sylvestris saplings (Korhonen et al. 1998). For H. parviporum, L. decidua extract had a significantly higher reduction effect on growth rate than P. abies and P. sylvestris extracts. Betula pubescens is occasionally attacked by both H. parviporum and H. annosum (Piri 1996; Lygis et al. 2004), and gave the weakest effect on growth rate for the two fungi. Heterobasidion spp. are known as aggressive fungi, easily attacking living trees. Hence, an obvious explanation of the AFA is that the white rot Heterobasidion species does not need to penetrate the bark to infect trees, the entry route is via roots or wounds (Woodward et al. 1998). Fomitopsis pinicola lives both parasitically and saprophytically on conifers and deciduous trees, but is most common on dead conifers, especially Picea and Pinus (Ryvarden and Gilbertson 1993). Among the conifer bark extracts, P. abies gave the highest AFA. Conifers are the main host, and P. abies is the most important wood substrate for F. pinicola in Norway. This can support that the fungus infect via roots or wounds, not through the bark.

Cancer fungi

The weakest effect of A. platanoides extract in this test was achieved for N. ditissima. Nectria ditissima infection seems to occur via leaf scars, small wounds caused by feeding insects and late frost injuries (Metzler et al. 2002) on deciduous trees. The related N. fuckeliana is a weak wood pathogen on conifers, as well as a common wound inhabiting species, causing necrotic bark canker (Langrell 2005). The coniferous bark extracts had a higher effect on N. ditissima than on N. fuckeliana. This might reflect a “host effect”. Phacidium coniferarum and N. fuckeliana were insignificantly affected by S. aucuparia extract, and with N. ditissima it had a slight positive effect on growth rate. On P. coniferarum with S. caprea and L. decidua, and on N. ditissima with S. aucuparia and B. pubescens, the bark extract amended plates showed significantly faster growth rates than the control. The positive effect of L. decidua even seemed to increase slightly with increasing concentration of the bark extract. No significant difference in growth response of P. coniferarum was found between P. abies and P. sylvestris.

Blue-stain fungi

Several species of fungi use the bark beetle Ips typographus L. as vector (e.g. Solheim 1986; Furniss et al. 1990), but the blue stain fungus C. polonica is the primary invader (Solheim 1992) and is alone able to kill healthy P. abies trees when mass inoculated (Krokene and Solheim 1998). Several of the bark extracts had a quite high AFA on C. polonica when compared to the other ascomycetes. For C. polonica, no significant difference was found between A. platanoides and A. incana extracts, and a high AFA was also found for P. abies extract. The generally high AFA effect on C. polonica might be explained by the use of the bark beetle vector between host trees. The fungi is not exposed to the bark extracts and hence do not need to produce any mechanisms to protect itself against the extracts.

Conclusions

This is the first report providing data on the AFA of several European tree species against some decay, blue-stain and canker fungi. The results show that there is no straight forward main host effect; the attack pattern is more complex. The study supports that bark is generally too effective to serve as an entry point for wood attacking fungi. The route of ingress of fungi gives better explanation of the results. The deciduous trees in our test were found to be less active against the canker and blue stain ascomycete fungi than the conifers. Generally the decay fungi were more inhibited by the bark extracts than the blue-stain fungi, while the lowest inhibition was found among the cancer fungi. Acer platanoides extract proved to be the most efficient bark extract tested, significantly reducing the growth rate of all tested fungi. Betula pubescens extract generally gave the weakest reduction in growth rate.