The presence of bacteria in birch trees invaded by the brown-rot

fungus Piptoporus betulinus

Research Paper 6 VWO, 2008-2009, on the occurrence of bacteria in birch trees colonized by birch polypore fungus

Corine Rewinkel Marnix College - Ede Jorien van der Wal 6 VWO, Cluster N&G, Topic Biology March 2009 Supervising teacher: Mrs. A. van Montfort

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ACKNOWLEDGEMENTS Lots of thanks to Prof. Wim van der Putten, head of the Department of Multitrophic Interactions, Centre for Terrestrial Ecology (CTE) of the Netherlands Institute of Ecology (NIOO), for offering the opportunity to execute this assignment at NIOO-CTE by forwarding our request to his colleague Dr. Wietse de Boer, senior researcher at the Department of Terrestrial Microbial Ecology at NIOO-CTE. Special thanks to Wietse the Boer, who made it possible to accomplish this assignment, for his enthusiastic guidance and assistance both in the field and in the laboratory and for all his explanations. Many thanks to Paulien Klein Gunnewiek and Wiecher Smant, technicians of the Department of Terrestrial Microbial Ecology who helped us with enumeration of bacteria and analyses of wood samples.

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TABLE OF CONTENTS Acknowledgements ..................................................................................................................... i Summary ...................................................................................................................................iii Our personal opinion on the research paper............................................................................... v 1 Introduction ........................................................................................................................ 1 2 Background information .................................................................................................... 3 2.1 Wood rot fungi ........................................................................................................... 3 2.2 Interaction between lignocellulose-degrading basidiomycetes and bacteria ............. 4 2.3 Results from a study on bacteria occurring in wood under decay by the white rot fungus Hypholoma fasciculare (sulphur tuft) by V. Valášková et al. (submitted) .................... 6 2.4 Research questions and hypothesis ............................................................................ 7 3 The set-up of our research.................................................................................................. 9 4 Sampling, sample preparation and laboratory analyses ................................................... 13 4.1 Wood sampling ........................................................................................................ 13 4.2 Preparation of the wood samples for further testing ................................................ 13 4.3 Wood analysis .......................................................................................................... 14

4.3.1 Moisture content and dry weight of the wood samples.................................... 14 4.3.2 pH and enzyme activity measurements........................................................... 14 4.3.3 Fungus-biomass determination ........................................................................ 15

4.4 Enumeration of bacteria ........................................................................................... 15 4.4.1 Preparation of the agar plates ........................................................................... 16 4.4.2 Extraction of the wood samples and inoculation of the agar plates ................. 16 4.4.3 Bacterial counts ................................................................................................ 17 4.4.4 DAPI stain for soil bacterial counts ................................................................. 17

5 Conclusions and discussion.............................................................................................. 23 6 Recommendations ............................................................................................................ 25 References ................................................................................................................................ 27 Annex 1 Research plan/plan of action ................................................................................ 29 Annex 2 Fungi..................................................................................................................... 33 Annex 3 DAPI-stain for soil bacterial counts (fluorescent staining) .................................. 35 Annex 4 PCR Detection of bacteria.................................................................................... 37

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SUMMARY A research paper is a compulsory secondary school graduation requirement. It has to be on one of the major subjects of your subject cluster or “profile”. As both of us took the N&G (Science and Health) cluster, we had the choice between doing a research paper on mathematics, chemistry, physics, or biology. We opted for the latter and for a topic on wood decomposition by fungi. We found Wietse de Boer of NIOO-CTE in Heteren willing to facilitate our study and supervise us. Upon further consultation the topic was made more specific and redefined as: The presence of bacteria in birch trees colonized by the brown-rot fungus, Piptoporus betulinus (birch polypore). The intention of the study was to gain more knowledge on the interactions between saprotrophic basidiomycetes and bacteria. This is not only important from a basic scientific point of view, but may also contribute to finding alternatives for wood impregnation and to the discovery of antibiotic metabolites. We took wood samples from birch trees colonised by the fungus P. betulinus in October 2008 from a location near the village of Doorwerth. The samples were analysed for moisture content, pH, enzyme activity, fungus biomass, and the presence of culturable bacteria Between October 2008 and March 2009 we visited the laboratory of Centre for Terrestrial Ecology (CTE) in Heteren several times to do the analyses. We found none or very low numbers of culturable bacteria in all our samples, in contradiction to the findings of a study on wood infested by white rot fungus Hypholoma fasciculare (Sulfur tuft) . The lack of bacteria in our wood samples confirmed an earlier incidental observation of P. betulinus in a study on H. fasciculare infested wood by Vendula Valášková. The lack of bacteria could not beforehand simply be attributed to P. betulinus having bactericidal effects/producing bactericidal metabolites (antibiotics). Other wood rot fungi like e.g. H. fasiculare also produce bactericidal metabolites (antibiotics) but still large numbers of cultivable bacteria were present in wood under decay by H. fasciculare. So, the absence or low abundance of bacteria in wood infested with P. betulinus could not have been predicted. Samples with the highest cellulase activity, i.e. the ones that are being decomposed at the highest rate, invariably have a low pH, indicating fungi cellulase functions well at low pH values, which again is a reason for the acidification of the environment by fungi. pH values varied strongly between the samples from very acid to mildly acid. There are no indications, however, that pH or acidification is a determining major factor for presence of bacteria, The large differences in enzyme activity between the samples can also not be linked to numbers of bacteria, and therefore also not to the presence of oxygen radicals that are linked to the lignin-degrading or -modifying enzymes. Thus, the production of reactive oxygen species as a bactericidal factor seems to be ruled out as well. Selection pressure of antibacterial metabolites of P. betulinus could have resulted in bacteria that adapted to the circumstances and became resistant, but this is not the case. For the time being, the only possible explanation for the virtual absence of bacteria, therefore, seems to be

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fungal production of toxic (bactericidal) substances. Perhaps toxic metabolites are being excreted by some fungi whereas others concentrate these in their fruiting bodies for protection. The findings of this study, i.e. the absence of culturable bacteria in wood decayed by a particular fungal species, are therefore new and interesting. More research will need to be done to fully explain the mechanisms involved and, as a next step, to find out which fungi have similar bacteria-suppressing effects and how fungi that suppress bacteria differ from the ones that don’t.

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OUR PERSONAL OPINION ON THE RESEARCH PAPER Corine During the process of finding a suitable topic, I never thought about the presence of bacteria in birch trees invaded by the brown-rot fungus Piptoporus betulinus. Jorien came with the idea of wood rot fungi, so we decided to contact NIOO-CTE to find out more about it. After we had decided on our topic, I still wasn’t sure what to expect of the study. The first day in the lab, I immediately got more interested. I enjoyed gaining lab experiences and I had fun in performing the experiments. It was really interesting to be able to see how a research is done and the multiple aspects that are involved. Time flew by while executing different experiments and procedures. In this way, I gained quite some experience in working with the microscope, proper treatment of samples, laboratory techniques, etc. Unfortunately, we don’t have the time to finish the research within the given time. It would have been nice to complete the entire experiment for our report. However, I was glad to participate in the fieldwork and lab work. Wietse de Boer was a great supervisor and he taught us a lot of new things about wood rot fungi. I never knew this subject could be so interesting, so I was really surprised. He and Paulien were very helpful in guiding us during our research. I also enjoyed working with Jorien, because we knew what to expect from each other and her enthusiasm during our trips to the institute was great. I’m really happy I was given this opportunity, even more because it was the first investigation towards interaction of brown rot fungi and bacteria. To conclude, I’m glad that we went to Heteren and got the chance to do some useful and enjoyable work. Jorien This research paper turned out to be a great experience. It was really nice to be offered the opportunity to work together with well-known researchers of NIOO-CTE, and to work on a very interesting topic on which more may become known in the coming years, upon further research. It was great to gain some more knowledge on wood rot fungi, on their role in nature, the possible interaction with bacteria, and it was great to get some experience with experiments and laboratory methods. I enjoyed the discussions with Wietse de Boer, our supervisor at NIOO-CTE. Although we started well in time with the preparations for our research paper and in contacting NIOO-CTE, and although we took the wood samples already in October, it proved to be complicated to finalize the assignment in the time set for the paper. This was mainly due to the tight work schedule we have at school in this last year, with many other assignments and school exams, as all visits to NIOO-CTE had to be planned during the school week. It was also due to the fact that we depended on the work schedule of the people at NIOO-CTE, apart from having to wait for bacteria to grow, before we could continue with the next phase.

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Actually, I think this is the only negative aspect in our research paper experience. Because of the time limit, we could not finalize everything we would have liked to do and could have accomplished, had we been allowed a few more weeks time to hand in the paper. Although, because of the time limit, it is now beyond our paper, we still plan to go to NIOO-CTE to observe a q-PCR procedure on our samples and we are very interested in the outcome. As on the outcomes of the study: I would have liked to see some bacteria growing on the plates, to be able to categorize them according to colour and shape, to study them further under the microscope, and to gain some experience in that too, but this is not what happened. However, the present outcome, no or only few bacteria in birch colonized by P. betulinus, may prove to be a very interesting one. I really enjoyed working with Corine on this assignment. It was nice to brainstorm with her on a possible topic, it was great fun to cycle with her to Heteren and do the lab experiments, and we worked well together in the report writing. All in all I can say that writing to NIOO-CTE has turned out a very good choice!

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1 INTRODUCTION A research paper is a requirement/ a compulsory part for the curriculum for the matriculation exam in 6 VWO. It should be on one of the major topics of your subject cluster. The two of us took the N&G (Science and Health) cluster and we opted for doing a research paper on a Biology topic. We thought it would be nice to do a research paper on a topic for which we could do some lab analyses on samples taken by ourselves, so that we could get some experience with the many aspects that are involved in research: sampling, laboratory analyses, data analysis or interpretation, literature review, and report writing. As it was autumn when we started thinking about a suitable topic, “mushrooms” came to our mind, then “mushrooms on tree trunks”, then “wood decay”, and the idea was born: Would it be possible to do something with wood decay and fungi? But, who could help us with this and where could we find proper laboratory facilities? Surfing on the internet for information on this topic brought us to the Centre for Terrestrial Ecology (CTE) of the Netherlands Institute for Ecology (NIOO-KNAW), which is located in Heteren, not very far from our school. NIOO-CTE does a lot of research on wood decay by fungi. We decided to contact the institute to see if they could help us. To our great surprise, we immediately received a very positive reply. We paid a first orientation visit to NIOO-CTE mid October 2008, to meet with researchers, to discuss with them the topic of fungal wood decay and to see what would be possible for us to do, within the time frame of our research paper. At NIOO-CTE, considerable research is being done on interactions between wood rot fungi and bacteria. Bacteria may have a strong impact on functioning of wood-degrading fungi and vice versa. Research on these interactions is not only important from a basic scientific point of view, but is also expected to yield possibilities for alternatives for wood impregnation to replace (now often banned) harmful chemicals and may lead to the discovery of metabolites with medical therapeutic value (de Boer and van der Wal, 2008, and Folman et al., 2008). In a recent study done at NIOO-CTE by a Czech guest researcher Vendula Valášková. on bacteria associated with wood colonised by the white rot fungus Hypholoma fasiculare (Sulphur tuft), very high bacterial numbers were found in wood under decay by this fungus (see section 2.3). We agreed that our research would focus on the presence of bacteria (both quantity and diversity) in birch trees (Betula spp.) that are infested by the birch polypore or razor-strop fungus Piptoporus betulinus1. So far, research at NIOO-CTE had concentrated on white rot fungi. White rot and brown rot are the most important types of fungal wood decay. As will be explained in section 2.1, there are some essential differences between white rot and brown rot decay. The birch

1 P. betulinus is a common pathogen on Birch trees that continues growing on dead trees, i.e. saprotrophic.

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polypore is a common brown rot fungus. Therefore, we selected this fungus as a representative to examine relationships between brown-rot fungi and bacteria. We would take wood samples from various infested birch trees and analyze the wood samples for numbers and types of bacteria. We would also use the same wood samples for measurements on fungal decay activities (enzyme assays2) and environmental parameters (pH). We would then be able to relate this to the number of bacteria present in the wood. The set-up appealed to us as it would enable us to get acquainted with a variety of laboratory techniques and to learn much about wood rot fungi and bacteria and their interaction This paper relates on our study, the study approach, and the findings. In Chapter 2 of this paper, we elaborate on wood rot fungi in general, on possible interactions between wood rot fungi and bacteria, and on findings from a recent study on bacteria associated with wood colonized by the white rot fungus Hypholoma fasciculare. Chapter 3 summarizes our research set-up. Chapter 4 is on the sampling and on the laboratory analyses. Chapter 5 discusses the findings and presents some conclusions, and Chapter 6 finishes the paper with some recommendations.

2 Enzyme assay: Laboratory method for measuring enzyme activity

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2 BACKGROUND INFORMATION

2.1 Wood rot fungi Wood decay fungi cause the wood in the centre of tree trunks and limbs to decay. Most wood decay fungi act on dead wood; they are real saprotrophs, e.g. H. fascuculare. Pathogenic wood decay fungi attack living trees. If conditions are favourable for the growth of the rot fungi, large part of the wood of living trees can decay in a relatively short time (months to years), due to the penetrating capacity of the hyphae of the fungi. Fungi that decay tree trunks and limbs are spread by airborne spores that infect trees through injuries and wounds. Some decay fungi infect the roots and can spread to nearby plants from the roots of infected hosts. Almost all species of woody plants may be affected by trunk and branch decay. Usually, wood decay is a disease of old, large trees. Both saprotrophic and pathogenic decay fungi destroy the plant’s internal supportive or structural components, cellulose, hemicellulose, and sometimes lignin. According to the type of decay, the fungi can be subdivided into three main groups: • White rot fungi decompose cellulose, hemicellulose and lignin and cause a

discolouration of the wood. The rotted wood feels moist, soft, and spongy, has fibrous or stringy appearance, and is pale in colour (white or yellow).

• Brown rot fungi mainly decay the cellulose and hemicellulose (carbohydrates) in wood, leaving behind the brown coloured lignin. Brown-rot fungi do modify lignin, however, as, to be able to get to the cellulose and hemi-cellulose (the real source of energy for the fungus). The lignin that protects (hemi)cellulose, needs to be degraded or modified (Fig. 1). Wood affected by brown rot is usually dry and fragile, and readily crumbles into cubes because of longitudinal and transverse cracks; it usually forms a solid column of rot in wood. Brown rot is generally more serious than white rot; infected wood may be greatly weakened.

• Soft rot is caused by both bacteria and fungi. They decay cellulose and hemicellulose, but they leave most lignin intact, but only in areas directly adjacent to their growth. The result is a softened surface layer. Soft rots grow more slowly than brown and white rots and usually do not cause extensive structural damage to wood of living trees.

Fig. 1 Overview woody cell wall

Fig. 2 White rot, brown rot and soft rot, respectively

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The white rot group of fungi is the largest and a very heterogeneous group. Only about 6% of wood-decay fungi cause brown rots. The majority of brown-rot and white-rot fungi belong to the Basidiomycetes. Soft-rot fungi belong to the Ascomycetes and Fungi Imperfecti, fungi that do not fit into the commonly established taxonomic classifications (see Annex 2 for more details on these taxonomic groups). Piptoporus betulinus is a brown rot fungus and belongs to the Basidiomycetes.

2.2 Interaction between lignocellulose-degrading basidiomycetes and bacteria Wood-inhabiting bacteria either originating from the tree, from the air, rain or soil, probably grow on easily degraded substrates as sugars, organic acids, pectin and easily accesses cellulose (Schmidt, 2006). Unlike the fungi, they have a very limited ability to degrade wood on their own as they have difficulties in penetrating solid substrates and are hardly able to degrade lignin. Some degrade parts of the lignified cell wall. Bacteria may play an important role in the functioning of lignocellulose-degrading basidiomycetes. They may have a negative effect on the fungal growth and fungal activity because they are competing for compounds that are released by extracellular fungal enzymes and sometimes fungi may be eaten by bacteria. Bacteria may also have a positive effect on fungal growth, through the supply of nitrogen and through detoxification of mycotoxic compounds. So, interaction between wood-decomposing basidiomycetes and bacteria can take various forms, ranging from predatory and competitive to mutualistic (i.e. benefiting both organisms). In the following part we try to summarize how these interactions function, though not all is known yet:

1. Competitive and antagonistic interactions

Basidiomycetes degrade lignocellulose-rich material and in doing so, they exude many different extracellular enzymes and small compounds (mediators) that are involved in the degradation of the different polymers. The oligomers released by the extracellular enzymes are the actual growth substrates for the fungi. These oligomers are also suitable substrates for most wood- and litter-inhabiting bacteria. Thus, bacteria may profit from the degradation activities of fungi (Fig. 3) . At the same time, this may deprive fungi of a considerable part of their growth substrates. To prevent this from happening, basidiomycetes may inhibit the growth of bacteria. This is found to be the case for the white-rot fungus Pleurotus ostreatus in both soil and straw (Lang et al., 1997; Gramms et al., 1999) and in timber blocks around basidiomycete mycelia(Gramms, 1987).

(LMW = Low Molecular Weight compounds)

Fig. 3 Schedule showing the dependence of bacteria on fungi

Extracellular fungal enzymes

Bacteria

LMW products

Fungi

Wood

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In a recent study Folman et al. (2008) examined the effects of two white-rot fungi, Hypholoma fasciculare and Resinicium bicolor on numbers of bacteria inhabiting beech wood blocks, and found that numbers of culturable3 wood-inhabiting bacteria and total bacteria (detected by microscopy) were considerably reduced after colonization of the blocks by the rot fungi. This points towards a bactericidal effect by the fungi. A follow-up study with H. fasciculare (de Boer et al., unpublished results), confirmed that the numbers of wood-inhabiting bacteria decreased already in the first weeks after colonization of the wood blocks by the fungus.

Mechanisms that may be involved in the killing of wood-inhabiting bacteria by basidiomycetes: • A rapid acidification of wood by the colonizing fungi via exudation of organic acids,

e.g. example oxalic acid. A rapid drop in pH is likely to be detrimental to many bacteria in particular when undissociated forms of weak organic acids are also present (Booth, 1985). These undissociated organic acids cross the bacterial cell membrane passively by diffusion and cause a fatal drop in the intracellular pH of the bacteria. Bacteria can adapt to low pH, but only when the pH decrease is gradual (De Boer et al., 1995).

• The production of reactive oxygen species by wood-decaying fungi, for example hydroxyl radicals4 that are involved in the modification or degradation of lignin may also be harmful to many bacteria. Bacteria surviving in wood colonized by decay fungi must have sufficient anti-oxidative activity to protect themselves from being attacked by radicals.

• Saprotrophic basidiomycetes produce secondary metabolites that may also inhibit or kill bacteria (Lorenzen and Anke, 1998; Abraham, 2001; Liu, 2005). Several basidiomycetes, e.g. Hypholoma fasciculare produce organohalogens, including chloroform, a biocidal compound (Hoekstra et al., 1998; Verhagen et al., 1998) that may suppress competing fungi and bacteria. Secondary metabolites (e.g. antibiotics) may also enhance the competitive position of bacteria against lignocellulolytic basidiomycetes. Strong suppression of the fungus, however, would in the end lead to starvation of the bacteria.(Fig. 3) Antagonism of bacteria against fungi appears to be more profitable during the initial stage of wood decay when easily degradable compounds are present.

2. Predation, parasitism and disease

Cellulolytic and lignolytic basiodiomycetes can penetrate bacterial colonies and then cause subsequent lysis of bacterial cells (e.g. P. ostreatus and Lentinula edodes, (Barron, 1988; Tsuneda and Thorn, 1994a; Barron, 2003). Nutrient-limiting conditions, viz. nitrogen limitation, seem to activate this behaviour of the fungi. The bacteria may be a valuable source of nitrogen for the fungi (Barron, 2003). Two white-rot basidiomycetes, H. fasciculare and R. bicolor, strongly reduced the number of wood-inhabiting bacteria upon colonization of beech wood blocks (Folman et al., 2008), probably due to fungal-induced lysis of the bacteria. Fungi can also be the victim of bacteria. So far, however, this has only been demonstrated in ascomycetes in an experimental setting. Bacterial pathogens on fruit bodies of saprotrophic basidiomycetes are a special case of mycophagy. The bacteria, when present in sufficient numbers, produce secondary metabolites that disrupt the fungal membrane,

3 Culturable bacteria: bacteria able to grow on a solid medium 4 Radical: an atom or group of atoms with one or more unpaired electrons. Radicals can have positive, negative or neutral charge. They are formed as intermediates in many biochemical reactions.

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and induce disease. The bacterium is thus growing at the expense of hyphal contents that are released when the fungal membranes disrupt (de Boer et al., 2005).

3. Mutualistic interactions

Litter- and wood-degrading basidiomycetes have developed special tactics for dealing with suboptimal concentrations of nitrogen in their substrates. These include recycling of nitrogen from aging mycelia, reallocation from intracellular stored proteins and uptake and translocation of nitrogen from soil to wood or litter (Cowling and Merrill, 1966; Watkinson et al., 2001; Lindahl and Finlay, 2005). As mentioned under point 2 above, lysis of bacteria may be a strategy for saprotrophic basidiomycetes to get hold of nitrogen (Greaves, 1971; Tsuneda and Thorn, 1994b). Nitrogen fixing bacteria that are adapted to grow in the vicinity of wood-degrading basidiomycetes may provide a continuous source of nitrogen to the fungi, that cannot fix nitrogen themselves. Bacteria may also attribute in other ways to mutualistic interactions with wood-decomposing basidiomycetes, by: • providing essential growth factors • degrading toxic compounds and so stimulating growth and activity of wood-degrading

fungi. (Fresh wood has several compounds, wood extractives, e.g. resin and tannins, that may inhibit growth and activity of basidiomycetes)

• detoxifying fungal cell membrane disrupting compounds that are produced by bacterial pathogens of fruit bodies

• removing fungal auto-inhibitors and thus enabling fruit body formation of several edible mushrooms (Noble et al., 2003) or alternatively exerting a stress on the fungus

that triggers fruit body formation.

2.3 Results from a study on bacteria occurring in wood under decay by the white rot fungus Hypholoma fasciculare (sulphur tuft) by V. Valášková et al. (submitted)

The species richness was estimated at around 120 and most bacteria were related to Proteobacteria and Acidobacteria (59%, resp 23%). The culturable part of the bacterial community consisted of acidophilic and acid-tolerant bacteria that rely on substrates that are released by lignocellulolytic enzyme activities of the fungus. No indications for antagonism (antibiosis5) of the bacteria against the fungus were detected. The reason for finding high numbers of bacteria in wood in an advanced stage of decay, contrary to fresh wood colonised by H. fasciculare, may be attributed to the fact that, H.

5 antagonistic association between an organism and the metabolic substances produced by another

Aim of the study was to investigate abundance, composition and properties of the bacterial community in wood degraded by the saprotrophic basidiomycete Hypholoma fasciculare. Based on the bactericidal effects that were found by Folman et al. (2008), it was expected that natural wood samples in a stage of advanced decay by H. fasciculare (present as fruiting bodies) would contain no or few bacteria. However, the wood samples (from a deciduous forest) were found to contain very high numbers of bacteria: 0.2-7.8 x 109 culturable bacteria per gram dry wood mass.

Fig. 4 H. Fasciculare

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fasciculare is an effective lignocellulose decomposer, changing the properties of colonized wood markedly. As wood degradation proceeds, suitable niches for bacteria may develop: • The breakdown of cellulose and hemicellulose results in smaller compounds (utilizable

sugars and organic acids), that are easily degradable for bacteria. Different metabolites were found to be used by different species of bacteria (different specific nutritional requirement) Also, wood in an advance stage of decay has a high fungal biomass content which is likely to be/produce suitable compounds for some species of bacteria.

• An environment with a low pH (3.6-4.3). Of the isolated, culturable bacteria, all but one were able to grow at pH 4.0, which indicates the species are acid-tolerant. pH seems to be a major factor defining community composition.

It seems that bacteria that are present in highly decayed wood colonised by H. fasciculare have adapted well to the environment, contrary to bacteria in freshly colonised wood. It is also possible that, when the decay advances, something changes in the behaviour of the fungus, and perhaps less toxic (bactericidal) exudates are produced.

2.4 Research questions and hypothesis The research question was: Are bacteria present in birch wood that is infested by the brown rot fungus Piptoporus betulinus as is the case in wood under decay by the white rot fungus Hypholoma fasciculare (see 2.3)? Are relations between brown rot fungi and bacteria similar to those between white rot fungi and bacteria? Are similar mechanisms involved? Based on the results of the study of V. Valášková et al., we expected many bacteria in the decaying birch wood samples.

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3 THE SET-UP OF OUR RESEARCH As mentioned in the introduction in Chapter 1, we planned to focus our research paper on the presence of bacteria, both quantity and diversity, in dead birch trees (Betula spp.) under decay by the birch polypore or razor-strop fungus, Piptoporus betulinus6, a brown rot fungus. It would be interesting to compare the results with those of research done at NIOO-CTE so far on the presence of bacteria in wood infected by white rot fungi. After the first meeting at NIOO-CTE with Wietse de Boer, a Plan of Action was prepared. The detailed plan is presented in Annex 1. In short, the approach was to take wood samples of five dead trees infested by P. betulinus, near young fruiting bodies7 of the fungus. Each tree would be sampled at two places at the trunk to find out whether there are differences between the two samples of one tree or between trees only. The total number of wood samples thus would amount to 5 x 2 = 10. In the lab, samples would be split into two subsamples, one for enzyme- and fungus biomass determinations, the other one for bacterial counts. Subsamples for enzyme measurements were to be kept in the freezer at a temperature of -20 ˚C. We would probably not be able to do the fungus enzyme and –biomass determinations ourselves as they are time-consuming, but they could be done by others at NIOO-CTE. Subsamples for bacterial counts would be stored at 4˚C and for this reason inoculation of the plates should start no more than two days after sampling. For practical reasons this procedure was slightly adapted. Because of our tight school schedule, we could not start work in the laboratory two days after sampling. The samples had to be kept for a bit longer and therefore they were stored in the freezer instead of being kept at 4˚C. To calculate the number of bacteria per gram dry wood as to compare the differences between trees, dry weight of the wood samples would be determined by overnight drying in a stove at 70˚C and subsequent weighing. For the bacterial counts we would use the procedure as described by Vendula Valášková who worked on bacteria in trees colonised by the white rot fungus Hypholoma fasciculare (sulphur tuft) Valášková et al., submitted); the procedure is described in detail in Annex 1 and in Chapter 5. This meant we would extract crushed wood samples with MES buffer, properly prepare the wood suspensions and four dilutions and then plating these in three different trypticase soy broth agar (at different pH values, i.e. for the selection of acidophilic bacteria), and one water yeast agar media. With 2 samples of each of the 5 trees, 4 dilutions, 2 repeats, and 4 growth media, the total number of agar plates would thus amount to 4 (dilutions) x 2 (repeats) x 10 (samples) x 4 (growth media) = 240. The dilutions we used were based on the results of the study Vendula Valášková.

6 P. betulinus is a common pathogen on Birch trees that continues growing on dead trees, i.e. saptrophic. 7 P. betulinus is annual, so we sampled near to new (active) fruiting bodies only

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The inoculated Petri dishes were to be incubated at 20°C for several weeks. After one week, and then several times in week 2, 3, and 4, the number of colony forming units (CFUs) would be observed. After week 1, growing bacteria in growth media TSB6.5 were to be categorized according to colony types into 10 different types, based on colony morphology, size, and colour. The counts would give an indication about fast- and slow-growing bacteria. Variety in colonies is an indicator of diversity. The number of bacteria per gram wood would be compared to the outcomes of Vendula Valášková’s study.

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Fig. 5 Tree sampling site, Doorwerth

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4 SAMPLING, SAMPLE PREPARATION AND LABORATORY ANALYSES

4.1 Wood sampling Wood samples were collected from birch trees at a forest site near Doorwerth village, from five randomly selected dead trees with Piptoporus betulinus. We selected trees that had at least two fresh fruiting bodies of Birch polypores each on their stems. Two of the five trees lay on the ground. For each sample, the chisel and tweezers were sterilized by pouring some alcohol and burning this using a lighter. Immediately above or beyond a fruiting body, a square of bark (approx. 4cm x 4cm) was removed from the tree with hammer and chisel. We also removed the outer layer (about 1 cm) of the wood to minimize contamination with bacteria from the outside of the trees. The chisel was then cleaned with alcohol (as described above) and forced deeper into the stem. We collected the wood sample using the pair of tweezers and filled a sterile plastic test tube with the wood fragments. The procedure was repeated for a second fruiting body on the same tree, and twice at four other trees.

4.2 Preparation of the wood samples for further testing Back in the NIOO laboratory, the 10 plastic test tubes with wood samples were weighed to 3 decimal places on an analytical balance. The material from each plastic test tube was deposited into a sterile Petri dish. In the laminar flow cabinet (sterile conditions), the wood was then cut/fragmented into smaller pieces with sterile Stanley knives and a pair of scissors, to get homogeneous samples. Cutting the wood in the fume hood took was time-consuming. Especially the wood that was not yet much decomposed was difficult to cut. We weighed a quantity of 0.5 gram of each crushed wood sample into a sterile test tube. The test tubes were subsequently put into the freezer at a temperature of -20 ˚C for storing until they could be further processed for inoculation of the agar plates for bacterial counts8. The remaining part of the sample was returned to the first test tube and stored at -20 ˚C. This residue was kept for pH determination, enzyme activity - an indicator of wood decay, and fungus biomass measurements. This could be at any time and would be done either by us, if time permitted, or by CTE researchers, if we would not be able to do it in the time set. Part of the wood samples was used to determine wood moisture content which is needed to be able to calculate the number of bacteria per gram dry wood as to compare the differences

8 The subsamples for the bacterial counts should have been stored at 4˚C no more than 2 days after sampling, but as this timing did not fit in the time schedule, they were stored at -20˚C, so that they could be kept for a longer period. It is assumed this did not affect bacterial life.

Materials used for taking wood samples: • lighter • plastic bottle/siphon with alcohol (75%) • 10 small, numbered plastic test tubes with caps • 20 cm long pair of tweezers • tissues • chisel • hammer

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between trees. For this determination, samples were weighed, then dried overnight in a stove at 70˚C, and weighed again.

4.3 Wood analysis

4.3.1 Moisture content and dry weight of the wood samples Part of the wood samples that were used for the bacterial counts, was meant to be used to determine dry weight. Dry weight is needed to be able to calculate the number of bacteria per gram dry wood as to compare the differences between trees. For this determination, samples were to be weighed, then dried overnight in a stove at 70˚C, and weighed again. We have not been able to do this, however, as the wood material that is left is still needed for DNA extraction. Once that is complete the moisture content determination will take place. This means that, for now, we can only express the results per fresh weight. We can estimate the moisture content at 80% (w/w), which is a good average in wood samples.

4.3.2 pH and enzyme activity measurements The pH measurements on the wood samples were done in water extracts (10 ml per gram fresh wood mass) after 2 hours of shaking. The enzyme data are represented here semi-quantitatively only, as otherwise we would have had to calculate with enzyme activity units9 and we would have had to present the elaborate protocols (assay methods). As we did not do the spectrophotometric analysis ourselves, we skipped this. Still, based on these data it is possible to say something about possible mechanisms involved: Table 1 shows that pH varies strongly between the samples. There are very acid samples, whereas others are much less acid (tree no. BII). As we did not detect bacteria in any of the samples, pH does not seem to be an explanative parameter, i.e. does not seem to be a major factor defining the occurrence of bacteria. The same is valid for the oxygen radicals that are linked to the lignin-degrading or -modifying enzymes (laccase and Mn-proxidase). There are large differences in enzyme activity between the samples, but this cannot be linked to numbers of bacteria. This means, that our outcomes do also not support the hypothesis of production of radicals as mentioned in section 2.2.

9 Amounts of enzymes can be expressed as molar amounts or measured in terms of activity, in enzyme units. Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. 1 Enzyme unit (EU) = 1 μmol min-1

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Table 1 Results of enzyme assays and pH measurements Sample no. Cellulase Laccase Mn-peroxidase pH B I * +++ - - 3.18 B II A - - + 4.60 B II B - - ++ 5.03 B III A + + +++ 4.65 B III B ++ ++ - 3.61 B IV A +++ + ++ 3.38 B IV B ++ +++ ++ 3.40 B V A +++ - + 3.42 B V B +++ - - 3.45

- no activity measured; + some activity measured, etc. Cellulase: mixture of enzymes that can degrade cellulose through hydrolysis Laccase: copper-containing oxidase enzymes that play a role in the degradation of lignin Mn-peroxidase: lignin degrading enzyme * Mixed sample as the individual quantities were too small for separate analysis, and to leave sufficient material for DNA extractions One could argue that the samples with the highest cellulase activity are being decomposed at the highest rate; these samples invariably have a low pH. This corresponds with the idea that fungi cellulases only function well at low pH values. This is one of the reasons that fungi acidify their environment. A high cellulase activity would also imply abundant nutrition for bacteria, but… they are not there! Possibly, the low pH values do still play a role in this. The results, however, could very well point towards the formation of toxic (bactericidal) substances. The results also show that there can be a large variation in properties of wood samples from a single tree as well as between trees.

4.3.3 Fungus-biomass determination Fungus-biomass determinations were mentioned in our original plan of action as a possibility but so far these results are not available. They may not become available either as most likely there is not enough left of the sample material as still some is needed to execute the DNA extraction.

4.4 Enumeration of bacteria The procedure for enumeration of bacteria was done as described by Vendula Valášková (Valášková et al., submitted). Her research was about bacteria in dead trees or stumps under decay by the white rot fungus Hypholoma fasciculare at NIOO-CTE. The procedure is described in detail in Annex 1.

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4.4.1 Preparation of the agar plates Three different growth media were prepared for the bacteria:

• 1/10 strength trypticase soy broth agar, pH 6.5 (TSB6.5) • 1/10 strength trypticase soy broth agar pH 5.0 (TSB5) - for the selection of acidophilic

bacteria • water yeast agar, pH 5.0 (WYA), a nutrient-poor medium for the selection of slow-

growing bacteria TSB media contains 1 g l-1 NaCl, 3 g l-1 trypticase soy broth (Oxoid), 20 g l-1 agar and either 1 g l-1 KH2PO4 (TSB6.5) or 1 .95 g l-1 MES (TSB5) as a buffering compound, and 0.1 g l-1 Delvocid (effective compound Natamycine, DSM Food Specialities) to prevent the growth of fungi. WYA contained 1 g l-1 NaCl, 0.1 g l-1 yeast extract (Difco Technical grade), 1.95 g l-1 MES, 20 g l-1 agar, and 0.1 g l-1 Delvocid. Before autoclaving for sterilisation, the pH was adjusted, using HCl and KOH. For preparation of pH 5.0 media, a double-strength agar suspension was autoclaved separately and mixed with media containing the other components after cooling to 55°C, in order to prevent liquefaction of the agar. A total of 240 agar plates was prepared.

4.4.2 Extraction of the wood samples and inoculation of the agar plates The whole procedure took place in the laminar flow cabinet to work in a sterile environment. To samples of ca. 0.5 gram fresh wood material we added 10 ml MES buffer (2-[N-morpholino]ethanesulphonic acid, 1.95 g l-1, pH 5.0, 10 ml per 1 g dry mass). The wood suspensions were shaken for 90 minutes on a vortex, then sonicated10 for 2 x 1 minute, and subsequently shaken for another 30 minutes. Dilutions were made in sterile MES pH5 buffer as follows: We filled just over 60 Eppendorf cups of 1.5 ml with 900 μl of MES pH5 buffer. The Eppendorf cups were inserted in a special rack/holder. A quantity of 100 μl of the sample fluid (the wood/MES buffer suspension; taken as 100) was pipetted into the first Eppendorf cup(10-1 solution). The cup was shaken on a vortex for a few seconds after which 100 μl was pipetted into the next Eppendorf cup (10-2 solution) . This was repeated 6 times (till a 10-6

solution). The first two dilutions were discarded. Quantities of 50 μl of the 10-3, 10-4, 10-5 and 10-6 solutions were plated in duplicate. We thus prepared 4 (dilutions) x 2 (repeats) x 10 (samples) = 80 agar plates per medium. With 4 media, this made a total of 240 plates. Approximately 10 small, sterile glass beads were applied to each agar plate with extract solution, shaken by hand for a few seconds to spread the solution evenly onto the agar plates, after which the glass beads were discarded. The agar plate was then covered with a lid and sealed with plastic tape.

10 Exposed to ultrasonic waves to loosen bacteria that adhere to wood particles

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The inoculated agar plates were incubated in a room at 20°C for a period of three weeks, with the intention to check the number of colony forming units (CFUs) on day 7 and several times in weeks 2 to 4. The growing bacteria were to be grouped into 10 different types, based on colony morphology.

4.4.3 Bacterial counts On day 7, none of the 240 agar plates had any signs of bacterial growth. The situation remained unchanged in weeks 2 and 3. In week 5 only one agar plate, plate no. TSB5 5B 10-3 showed some bacterial growth. Upon this result, it was decided to also verify microscopically whether numbers of bacteria were actually low in the wood samples, whether they were no artefact because of the inability of the bacteria to grow on the media used. We have done this by executing a DAPI stain procedure on some of the samples from the birch trees, on one wood sample from Fagus sylvatica of which it was known it was infested with bacteria (positive control), and on the bacteria that grew on plate number TSB5 5B 10-3. The procedure is described in the next section. Additionally, we took at random 6 agar plates and placed them under the microscope to verify whether any microcolonies of bacteria could be observed. The result was negative.

4.4.4 DAPI stain for soil bacterial counts

DAPI or (2,4-)Diamino diphenyl-indole is a fluorescent stain that binds strongly to DNA. It is used extensively in fluorescence microscopy. DAPI will pass through intact cell membranes, so it may be used to stain both live and fixed cells. For fluorescence microscopy, DAPI is excited with ultraviolet light. The emission appears blue/cyan. More details are in Annex 3.

We weighed 0.2 gram of fresh woody material of tree 1, tree 3 and tree 5, and 0.2 gram of a wood sample from a beech tree (Fagus sylvatica) that was known to have bacteria, into plastic test tubes with lids. A quantity of 5 ml demi water was added to the wood samples. We also took material from the only Petri dish that showed occurrence of bacteria (circular colony shape), TSB5 5B 10-3, with a glass rod. The test tubes were subsequently shaken in a shaking machine for 90 minutes.

Table 2 Samples for DAPI stain test

Sample no. Origin Sample weight

1 Birch tree no. 1 0.2 gram fresh wood 2 Birch tree no. 3 0.2 gram fresh wood 3 Birch tree no. 5 0.2 gram fresh wood 4 Beech tree 0.2 gram fresh wood 5 plate no. TSB5 5B 10-3 Small quantity scraped off agar surface

In the mean time we took 4 teflon-coated microscope slides, with 3 wells each. The wells of two slides were coated with a thin layer (drop) of liquid soap for optimal dispersion of bacteria; the other two slides were left untouched.

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With a pipette we applied a small quantity of extract from birch trees 1, 3 and 5 to the respective wells of one slide with detergent, and to the wells of one slide without detergent. The beech extract was applied to two wells of the second slide with detergent and to two wells of the second slide without detergent. A glass rod was used to remove bacteria from agar plate no. TSB5 5B 10-3 and to apply to the last well covered with detergent and the one without detergent.

with detergent with detergent

without detergent without detergent Fig. 6 Sequence of extracts for DAPI stain procedure (numbers corresponding

with the ones in Table 2) The slides were left to dry in the flow cabinet. When dry, they were fixed above a flame. 5 ml demi water was mixed with 10 µl DAPI solution in a plastic tube with a lid and shaken on a vortex. The tube was then covered with aluminium foil to prevent fading of the colour as DAPI is light sensitive. In the mean time we took at random 6 agar plates and placed them under the microscope to see if we could see any bacteria. The result proved to be negative. 40 µl of the diluted DAPI solution was added to each of the wells by means of a pipette. The slides were left in the dark (in a polystyrene box with a lid) for 10 minutes for the stain to be absorbed well/or; for the samples to stain. The slides were then removed from the box with tweezers and washed in three glass beakers filled with demi water to remove any excessive stain. The slides were then kept between tissue paper in a covered polystyrene box. To each slide, a few drops of anti-fading solution (glycerol and vitamin C) were added with a pipette between the wells. Subsequently, the slides were covered with a cover glass so that the anti-fading solution spread over the entire surface of the slide, and the cover glass was mounted using nail polish. When dry, each slide was studied under the fluorescence microscope. The DAPI stain colours the DNA of the bacteria blue (see Annex 3). However, also wood fibres are coloured blue by the DAPI stain. They can partly be distinguished by the eye (see Fig. 7). All other material that is not coloured by the stain appears blackish or brownish.

1 2 3 4 4 5

1 2 3 4 4 5

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Observations In the beech sample (well numbers 4) relatively many bacteria could be distinguished. Wells number 5 also showed blue colouring of the bacteria. Well numbers 1, 2 and 3 showed some blue colouring but this may also be attributed to wood fibres and fungus hyphen (especially in well number 3). To be able to really distinguish DNA from wood fibres, a quantitative PCR enumeration will have to be executed. The colouring was observed slightly better on the slides of which the wells had not been coated with detergent. Apparently, the DAPT stain also colours the liquid soap slightly, which leads to less contrast with the background.

Fig. 7 Dapi-stained bacteria and wood fibres under microscope

All in all, it is reasonable to conclude the DAPI stain confirmed the finding of the plates and that numbers of bacteria in the birch samples are really low. Unfortunately, the PCR procedure did not fit anymore within the time frame of our study as the research paper had to be handed in early March, so we were not able to say whether the observations in wells 1, 2 and 3 were really attributable to the presence of bacteria or whether they were artefacts, small particles of wood that also were coloured blue by the DAPI stain. The PCR will still be executed by researchers at NIOO-CTE, however, and we will be eager to go and follow the procedure and the outcome if it happens within the coming months. As the low numbers of bacteria may be partly due to contamination of the surface of the wood and as the activity or interaction of these low numbers of bacteria with the fungus can never be much, the bacteria were not further studied in detail.

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Fig 8 Preparing wood extraxts and inoculation of different growth media

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Fig. 9 Performing DAPI stain procedure

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5 CONCLUSIONS AND DISCUSSION All five P. betulinus infested birch trees sampled showed no or very low numbers of culturable bacteria. In comparison with the H. fasciculare infested wood samples of the study of V. Valášková, this seems to be a very different finding. We will have to be a bit careful in concluding this as, in our study, we opted for using the same dilutions as were used in the study on bacteria in wood colonized by the white rot fungus Hypholoma fasciculare by V. Valášková et al. The numbers of culturable bacteria in that study were found to be very high, viz. 0.2 -7.8 x 109 per gram dry wood material. With the dilutions we used, one can calculate the lower limit that we could have counted:

We plated dilutions 10-3 and up. This implies a lower detection limit of 5 × 105 bacteria g-1. The calculation is based on 1 bacterium at a dilution of 10-3. This is from 50 µl solution, so for the 10 ml suspension this has to be multiplied with a factor 2 × 105. This is for 0.5 gram wood, so for 1 gram it has to be multiplied with a factor 2 again. As this is based on fresh weight, the outcome for dry wood, will be slighter higher, about 5 × 105 g-1g fresh wood material

So, it is possible that lower numbers of bacteria may have been present in our samples, but could not be detected. Had we known beforehand that we would find no/very low bacteria numbers, we could have better plated less diluted MES solutions, but we anticipated much higher numbers of bacteria. Still, the lower detection limit of 5 × 105 is very low as compared to the large numbers found in the H. fasciculare study: 0.2 -7.8 x 109 per gram dry wood material. It seems thus correct to state that our study proves that P. betulinus infested birch wood had only few bacteria. In Valášková’s study, she also sampled one lying birch tree trunk that was colonised by P. betulinus and this sample was also found to have very low amounts of bacteria (Wietse de Boer, personal communication, December 2008). This incidental observation seems now to be confirmed by our results. One could wonder what the reason of the low numbers of bacteria can be: Is this a general difference between white rot and brown rot fungi or is the difference determined by the specific type of fungus? Further research is definitively needed on this. There are essential differences between white rot and brown rot fungi (section 2.1). White rot fungi decompose lignin, whereas brown rot fungi do not. Brown-rot fungi, however, do modify lignin, i.e. they chemically alter it in a way that enzymes that decompose cellulose and hemi-cellulose can penetrate the wood cell walls. This means there is a possibility that the accessibility of wood to bacteria is higher in the presence of white rot fungi than in the presence of brown rot fungi. Within the time frame of our study it was impossible to investigate the exact mechanisms involved. But, based on our results, we can conclude something on the mechanisms (see (section 2.2, under point 1):

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Samples with the highest cellulase activity, i.e. the ones that are being decomposed at the highest rate, invariably have a low pH, indicating fungal cellulase functions well at low pH values, which again is a reason for the acidification of the environment by fungi. pH values vary strongly between the samples from very acid to mildly acid. There are no indications, however, that pH or acidification is a determining major factor for presence of bacteria. The large differences in enzyme activity between the samples can also not be linked to numbers of bacteria, and therefore also not the presence of oxygen radicals that are linked to the lignin-degrading or -modifying enzymes. Thus, the production of reactive oxygen species as a determining factor seems to be ruled out as well. It could well be that toxic substances with a bactericidal effect are involved. As this is most interesting to find out, researchers at CTE will certainly continue to investigate this in the near future. It might well be that this leads to the discovery of new antibiotics. When no bacteria were found, we wondered whether perhaps we could have expected to find no bacteria in wood infested with P. betulinus, as this fungus is known to have antibacterial/bactericidal effects. Ötzi the Iceman, who appeared from a glacier in the Ötztal Alps, Austria in 1991, were he had been since 3300 BC, had P. betulinus in his pocket to fight off bacterial infections and there are also several publications of studies that show that P. betulinus produces bactericidal metabolites (antibiotics). We discussed this with our supervisor at NIOO-CTE, Wietse de Boer, who argued that this argumentation is not likely to be valid. Also Hypholoma fasiculare produces bactericidal metabolites (antibiotics) and has a strong bactericidal effect on bacteria that are already present in wood upon its colonisation, but still in the study on wood colonized by Hypholoma fasciculare large numbers of bacteria were found. Apart from this, tests on antibacterial effects are generally done in search for new antibiotics; they are always done “in vitro”, i.e. on plates or in liquid media with bacteria that are in most case human pathogens. These are bacteria that are not very common in combination with wood-degrading fungi. Growth arrest of such bacteria doesn’t say anything about the occurrence or absence of bacteria in natural wood samples colonized by P. betulinus. One could also argue that selection pressure of antibacterial metabolites of P. betulinus could have resulted in bacteria that adapted to the circumstances (like the many species of bacteria that became resistant to antibiotics due to high selection pressure because of the extensive use of antibiotics). This seems not to be the case, which is somewhat peculiar. For the time being, however, the only explanation for the virtual absence of bacteria seems to be a possible fungal production of toxic substances. Perhaps toxic metabolites are being excreted by some fungi, whereas others concentrate them in their fruiting bodies for protection. All this implies that the absence of bacteria in wood infested with P. betulinus could not have been predicted and that the findings of this study are new and interesting. The question that follows is: Which fungi have similar bacteria-suppressing effects and how do fungi that suppress bacteria differ from the ones that do not do so?

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6 RECOMMENDATIONS Research on interactions between various wood rot fungi and bacteria will certainly continue at NIOO-CTE. With the experience of our study it seems to be reasonable to advise to go, in first instance, for quantitative PCR assay for detection and enumeration of bacteria. Plating extractions on growth media to obtain colony forming bacteria should only happen if there are actual indications for the presence of bacteria. This will avoid having to prepare huge numbers of plates for perhaps little result. MES plating solutions could best be less diluted than the 10-3 and up solutions we used, in order to decrease the lower detection limit. We conclude with some very practical tips: • For reaching places on tree trunks near to fruiting bodies, a ladder is an essential tool. • Wood samples may be better taken from tree trunks using an electrical drill, as it will

provide much finer material than samples taken by hammer and chisel that still need thorough fragmenting before the fibres can be processed further.

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REFERENCES Barron, G. L. 2003. Fungi and the carbon cycle, from: Predatory fungi, wood decay, and the carbon cycle. Biodiversity 4: 3-9. Benko, R. Highley, T.L. 1990. Biological Control of Wood-Attacking Fungi Using Bacteria, Biodeterioration Research 3: 327-332 De Boer, W. and Van der Wal, A. 2008. Interactions between Saprotrophic Basidiomycetes and Bacteria, pp. 141-151, Chapter 8 from Ecology of Saprotrophic Basidiomycetes, by Lynne Boddy, Juliet Frankland, Pieter Van West, British Mycological Society Series, Academic Press De Boer, W., Folman, L.B., Summerbell, R.C., Boddy, L. 2005. Living in a fungal world: impact of fungi on soil bacterial niche development, FEMS Microbiology Reviews 29: 795–811. Deacon J.2006. Armillaria mellea and other wood-decay fungi, from The Microbial World, microorganisms and microbial activities: Institute of Cell and Molecular Biology, University of Edinburgh, http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/armill.htm Deacon, J. 2005. Wood decay, and wood-rotting fungi, from textbook Fungal Biology, Blackwell Publishing, (http://www.biology.ed.ac.uk/research/groups/jdeacon/FungalBiology/woodrots.htm) Folman, L.B., Klein Gunnewiek, P.J.A., Boddy, L. & de Boer, W. 2007. Impact of white-rot fungi on numbers and community composition of bacteria colonizing beech wood from forest soil. FEMS Microbiol Ecol 63:181-191. Highley, T.L. and Illman, B.L. 1991. Progress in understanding how brown-rot fungi degrade cellulose, Biodeterioration Abstracts, , Vol 5, no 3, pp. 231-244 Lepp, H. Fungal ecology, Wood rotting fungi, Australian fungi website. Updated on web 13 December, 2005 by Murray Fagg (anbg-info@anbg.gov.au) Valášková, V., de Boer, W., Klein Gunnewiek, P., Pospíšek, M., Baldrian, P.. Bacteria associated with wood colonized by the white rot fungus Hypholoma fasciculare: phylogenetic composition and properties. ISME Journal (submitted) Valá ková, V. and Baldrian, P. 2006. Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus – production of extracellular enzymes and characterization of the major cellulases, in Microbiology 152 (2006), 3613-3622; DOI 10.1099/mic.0.29149-0 Van der Wal, A., de Boer, W., Smant, W., van Veen, J.A. 2007. Initial decay of woody fragments in soil is influenced by size, vertical position, nitrogen availability and soil origin, Plant Soil 301:189–201.

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Web sources: Wood Decay Fungi in Landscape Trees, 2003, http://www.ipm.ucdavis.edu/PMG/PESTNOTES/pn74109.html Forest and Shade Tree Pathology, Wood Decay, from http://www.forestpathology.org/decay.html, College of Environmental Science and Forestry Enzymology of wood decay, from http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/armill.htm Microbes in trees and wood, University of Minnesota, web site for Diseases of Forest and Shade Trees Introductory Mycology BOT 461/561, Powerpoint presentation, from Northwest Alliance for Computational Science & Engineering (NACSE), Oregon State University, 2006, http://ocid.nacse.org/classroom/fungi/bot461/syllabus.htm Autoclaving Instructions, http://www.microbiol.unimelb.edu.au/staff/facilities/autoclave.html Polymerase chain reaction, Wikipedia, http://en.wikipedia.org/wiki/polymerase_chain_reaction Principle of the PCR, 1999, http://users.ugent.be/~avierstr/principles/pcr.html Zo werkt PCR, http://www.watisgenomics.nl/genomics/genomics/i000835.html: Wood rotting fungi, nematodes and bacteria, http://www.uoguelph.ca/~gbarron/MISC2003/feb03.htm Wood decay and forest disease, Powerpoint presentation, Northwest Alliance for Computational Science & Engineering (NACSE), Oregon State University, 2006. http://ocid.nacse.org/classroom/fungi/bot461/lectures/Lecture%207.ppt

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ANNEX 1 RESEARCH PLAN/PLAN OF ACTION SAMPLING We will sample 5 trees. Remove bark with a Stanley knife and also remove upper 1-cm layer of wood. Take a sample of the wood with a hand wood drill or with battery powered drill till a depth of 5 cm. Collect the material in a sterile plastic bag. Note that the metal drill bits need to be sterilised beforehand in the lab. Sterilising the drill bits in the field before each new sampling will be done using alcohol. Each tree will be sampled at two places to find out whether there are differences between the two samples of one tree or between trees only. Total number of samples: 5 x 2 = 10. List of necessary items • Stanley knives with several blades • Hand or battery-powered drill • Sterile plastic bags (at least 10) • Alcohol for sterilising drill bits and cotton wool (?) LABORATORY In the lab all samples will be split into two subsamples. One subsample will be used for enzyme- and fungus biomass determinations/measurements, the other one for bacterial counts. • Subsamples for enzyme measurements will be kept in the freezer at a temperature of -20 ˚C.

• Subsamples for the bacteria counts will be stored at 4˚C and for this reason the counts should be started no more than 2 days after sampling.

Counts Part of the wood samples that are meant for the counts, will be used to determine dry weight which is needed to be able to calculate the number of bacteria per gram dry wood as to compare the differences between trees. For this determination, samples are being weighed, then dried overnight in a stove at 70˚C, and weighed again after drying. The counts will be done as described by Vendula Valášková who worked on bacteria in trees colonised by the white rot fungus Hypholoma fasciculare (sulphur tuft) at NIOO:

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Materials necessary for the counts Based on the method in the textbox above, we need the following materials: • Sterile MES buffer: 3 x 50 ml • Sterile Eppendorf cups for preparing diluted solutions

• Sterile pipet tips (1ml and 0.2 ml) for making dilutions and inoculating the plates

• Sterile glass beads for spreading bacteria on the agar plates

• 3 different agar media (pH 6.5 (TSB6.5), pH 5.0 (TSB5) and water yeast agar, pH 5.0 (WYA)

• Number of agar plates necessary: 4 (dilutions) x 2 (repeats) x 10 (samples) = 80 agar plates per

medium (240 plates)

• Dilutions (based on experiences of Vendula): 10-3 to 10-6 in which

the first suspension (wood in MES buffer) is taken as a 100 dilution

• Samples of fresh milled wood were extracted with MES buffer (2-[N-

morpholino]ethanesulphonic acid, 1.95 g l-1, pH 5.0, 10 ml per 1 g dry mass).

• The wood suspensions were shaken for 90 min at laboratory temperature on a vortex, sonicated for 2 × 30 s and subsequently shaken for another 30 min using a vortex as described previously (Folman et al., 2008).

• Dilutions were prepared in sterile MES buffer and 50 μl aliquots of the dilutions were plated in duplicate.

• Three different media [1/10 strength trypticase soy broth agar, pH 6.5 (TSB6.5) and pH 5.0 (TSB5) for the selection of acidophilic bacteria as well as water yeast agar, pH 5.0 (WYA)] were used for plating. TSB media contained 1 g l-1 NaCl, 3 g l-1 trypticase soy broth (Oxoid), 20 g l-1 agar and either 1 g l-1 KH2PO4 (TSB6) or 1.95 g l-1 MES (TSB5) as a buffering compound and 0.1 g l-1 Delvocid (effective compound Natamycine, DSM Food Specialities) to prevent the growth of fungi. WYA contained 1 g l-1 NaCl, 0.1 g l-1 yeast extract (Difco Technical grade), 1.95 g l-1 MES, 20 g l-1 agar, and 0.1 g l-1

Delvocid. • Before autoclaving, the pH was adjusted using HCl and KOH. For

preparation of media of pH 5.0, a double-strength agar suspension was autoclaved separately and mixed with media containing the other components after cooling to 55°C, in order to prevent liquefaction of the agar.

• Inoculated Petri dishes were incubated at 20°C for 2 weeks. The number of colony forming units (CFUs) was determined by observation on day 7. Growing bacteria were grouped into 10 different types based on colony morphology.

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Actual counts and observations The number of bacteria per gram wood will be calculated and compared to the outcomes of Vendula’s experiments After one week, bacteria inoculated in the pH 6.5 (TSB6.5) medium will be categorized according to colony type and described (e.g. white/smooth/diameter 0.3 – 0.5 mm). The variety in colonies is a n indicator of diversity. We should also compare these results with those of Vendula’s experiment. Fungus enzyme and fungus biomass measurements are interesting to do to see how much fungi are available and how active they are. This may then be related to the numbers of bacteria. As both procedures require considerable time, we will not be able to do these ourselves alone in the time we have for this assignment. They may be done by others at NIOO-CTE, however.

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ANNEX 2 FUNGI Fungi are eukaryotic, spore bearing, heterotropic organisms that produce extracellular enzymes and absorb their nutrition. They differ in the way they take their nutrition. Saprobes for example, decay dead organic matter. There are several phyla (basal lineages) of fungi. Among the largest phyla are the Ascomycota and the Basidiomycota. Ascomycota or cup or sac fungi The Ascomycota or Ascomycetes, constitute the largest taxonomic group within the Eumycota. These fungi form meiotic spores called ascospores, which are enclosed in a special sac-like structure called an ascus. This division includes morels, a few mushrooms and truffles, single-celled yeasts (e.g., of the genera Saccharomyces, Kluyveromyces, Pichia, and Candida), and many filamentous fungi living as saprotrophs, parasites, and mutualistic symbionts. Prominent and important genera of filamentous ascomycetes include Aspergillus, Fusarium, and Claviceps. Ascomycota are characterized by a cup-like sexual reproductive structure and the production of a series of sacks, each called an ascus Basidiomycota or club fungi Members of the Basidiomycota, basidiomycetes, produce meiospores called basidiospores on club-like stalks called basidia. Most common mushrooms belong to this group, as well as rust and smut fungi, (Mushrooms, rusts, smuts, etc.). The Basidiomycetes are the familiar mushrooms, although there are several species of Basidiomycetes that would not be immediately recognizable as mushrooms such as the rusts and smuts. Sexual reproduction in the Basidiomycetes produces a structure with the familiar mushroom shape and results in haploid spores called Basidia.

Formation of Basidiospores Fungi imperfectae or Deuteromycota: a miscellaneous group of species for which sexual reproductive structures are unknown and therefore cannot be assign to any other group. An example is Pencillium.

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ANNEX 3 DAPI-STAIN FOR SOIL BACTERIAL COUNTS (FLUORESCENT STAINING)11 Solutions needed - 96% ethanol - soap (Dreft) - SDW (sterile demineralized water) - DAPI [(2,4-)Diamino diphenyl-indole]: 0.002 g l-1 prepare from stock solution 1 g l-1 (in freezer -20°C pm-lab ready-to-use portions) add 20 µl stock to 10 ml SDW - Anti-fading solution12: glycerol:PBS 13(1:1) with 0.5% ascorbinezuur (=anti-oxidant)

(PBS= 10 mM NaPO4 + 130 mM NaCl, pH7.5) Material needed Pipets (5 ml, 1 ml, 200, 20 µl) Coated slides (Nutacon TEFLON coating, 10-well 8 mm Nutacon , Staining trays + slide-racks, material to cover the trays for dark incubation Cover slides Nail polish Procedure

- Add 5 gram of soil (dry weight basis) to 50 ml sterile 0.25 g/l KH2PO4 (no pH adjustment or pH 6.5) in 100 ml Erlenmeyer flasks

- Shake 90 min on rotary shaker (150 rpm), sonicate for 2 min., and shake again for 30 min.

- Dilute soil suspension 10 x with sterile demi water (not when bacterial numbers are expected to be low e.g. in organic-poor soils)

- Use (diluted) soil suspension for counting immediately or store at 4 ˚C after addition of 0.1 ml 38% formaldehyde to 1 ml of suspension (maximum storage time 3 weeks).

- Coat wells of a Nutacon slide with a thin film of soap (Dreft). This for a equal dispersion of bacteria and soil particles.

- Add 5 μl of soil suspension to the coated wells of the Nutacon slide (at least 2 replicates per soil suspension)

- Dry at room temp. (when a lot of organic matter is present, dry for 2 h at 50°C) - Fix in flame - Put slides in a plastic box or large Petri-dish with wet tissues (from now on, work in

dark as much as possible) - Add 5 μl DAPI solution (2 μg ml-1 in demi water) per well. - Stain 5-10 min while covering the box or Petri-dish to prevent drying - Remove stain first with filter paper

11 Note: This description highlights the principle of the technique. It is method to specifically colour soil bacteria. We adapted the method accordingly. 12 The main purpose of anti-fading solution is to reduce the dye photobleaching rate, giving researchers longer observation time 13 PBS: Phosphate-buffered saline, a biological buffer that provides exactly what its name implies: a buffer to maintain a constant pH stays approximately constant) and just as many ions per unit volume as the inside of a cell (so that the cells don't swell or shrink); it contains sodium chloride, sodium phosphate, sometimes potassium chloride and potassium phosphate.

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- Rinse 3 times with SDW and dry slides in the dark (room temperature). - Add a drop of glycerol/antifading solution to each well - Cover with cover-slide and seal with nail polish - Count at 10 x 100 at UV illumination and the appropriate filter set. - Calculation: TBC= D x B x M x W-1

TBC= total bacteria count per g soil (cells g-1) D= 200 (in case of 5 μl ) * 10 (only if 10-fold diluted suspension has been used) * 50 ( = 50 ml suspension) / 5 (5 gram soil.) To be precise 50 should be replaced by 50+x (x ml moist in soil sample), but this will only give slight differences for soil samples with a high moisture content = organic layers B= mean count of bacteria per counting area M= microscopic factor (well area/ area of counting field) W= weight of ovendry soil sample (g)

Note: The DAPI dye binds with DNA. Therefore, these bacterial counts represent both the dead (provided that the DNA is intact) and the living microbial population.

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ANNEX 4 PCR DETECTION OF BACTERIA (This description of a general PCR is only presented to complete the picture. The actual quantitative PCR that will be effectuated on the wood samples will be slightly different) PCR stands for Polymerase Chain Reaction. It is a technique to copy DNA, actually a method by which a few fragments of DNA can be duplicated into millions in a couple of hours. The technique was developed by Kary Mullis in 1984 (he was awarded the Nobel Prize in Chemistry for it in 1993) and it has become a common technique in medical and biological research labs. PCR is used for various applications, including identification of genetic fingerprints, DNA cloning for sequencing and the diagnosis of hereditary or infectious diseases. Specific regions of a DNA strand are amplified. This is done with polymerase, primers, deoxynucleotide triphosphates (the building blocks from which the DNA polymerases synthesize a new DNA strand), a buffer, and Mg or Mn and K ions. The PCR usually consists of a series of 20 to 40 repeated temperature changes or cycles; each cycle has 3 discrete temperature steps:

1. Exponential amplification, where the amount of product is doubled each cycle. 2. Levelling-off stage. Here the DNA polymerase loses its activity and the use of

reagents like primers both causes the reaction to slow down. 3. Plateau stage, in which no new products are gained because of exhaustion of the

reagents.

Each cycle is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of dNTPs (deoxyribonucleotide trophosphate, the building blocks of DNA) in the reaction, and the melting temperature of the primers.

There are 5 phases: • Initialization step, 94-96°C (or 98°C if extremely thermostable polymerases are used)

for 1-9 minutes • Denaturation step, heating the reaction to 94-98°C for 20-30 seconds. The DNA

template and primers are melted by breaking the hydrogen bonds between complementary bases of the DNA strands. The DNA disentangles and single strands of DNA are the result.

• Annealing14 step, temperature is lowered to 50-65°C for 20-40 seconds. This allows annealing (attaching) of the primers to the single-stranded DNA template. The hydrogen bonds of stable DNA-DNA can only be formed when the primer sequence closely matches the template sequence. The DNA synthesis starts when the polymerase binds to the primer-template hybrid.

• Extension/elongation step, the temperature at this step depends on the enzym, the DNA polymerase, used. The DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding the building blocks dNTPs, resulting in a double strand of DNA again. The extension time depends on the length

14 Anneal: harden by heat treatment

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of the DNA fragment to be amplified and on the DNA polymerase used. Under optimum conditions the amount of DNA target is doubled at each extension step.

• Final elongation step, performed at a

temperature of 70-74°C for 5-15 minutes. This makes sure that any remaining single-stranded DNA is completely extended.

• Final hold, at 4-15°C, for an indefinite time; is for short-term storage of the reaction.

The PCR is mostly done in a reaction volume of 10-200 μl in small reaction tubes of 0.2-0.5 ml volumes. The process is carried out in a thermal cycler, meaning that it cools and heats the reaction tubes to achieve the right temperature at each step of the reaction.

The length of time and the used temperatures in each cycle depend on various parameters,

Schematic drawing of the PCR cycle

Explanation to drawing: Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which then are used as templates. Temperature steps: 1. Denaturing at 94-96°C 2. Annealing at 65°C 3. Elongation at 72°C