mycologist 20 (2006) 140143

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On the ecology of fungal consortia of spring sap-ows

Roland W. S. WEBER
Department of Biotechnology, University of Kaiserslautern, Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern, Germany

abstract
Keywords: Betula Biolm Consortium Insect vectors Mycocins Sap-ow Trichosporon pullulans Xanthophyllomyces dendrorhous Yeasts Spring sap-ow, i.e. xylem sap exuding through wounds of deciduous trees in spring, quickly becomes colonised by yeasts and lamentous fungi, of which several species occur regularly in consortia from different trees or regions. This article considers the survival, spread and population dynamics of these fungi, with particular emphasis on the substantial research by Wladyslav I. Golubev on birch sap-ows in Russia. Most fungi survive in the soil or epiphytically on tree trunks, and dispersal between trees is probably mediated by insects. Competition within and between sap-ow yeast species may be due to nutritional effects or the action of killer toxins (mycocins). 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

In the May issue of this years Mycologist I co-authored a short article describing a colourful consortium of lamentous fungi and yeasts colonising freshly cut birch stumps in Germany (Weber et al. 2006). Despite substantial literature searches on this topic, I remained unaware of a series of three seminal and highly relevant papers published by Wladyslav I. Golubev in the former Soviet Union (Golubev et al. 1977a,b, Shkidchenko & Golubev 1980). Although these articles are available in English translations, they have been cited only rarely by non-Russian scientists. It is therefore appropriate to discuss Golubevs work here, not least because it provides an interesting background and contrast to our results on red yeast consortia discovered in Germany (Weber et al. 2006) and subsequently in Italy (Weber & Davoli 2005).

2.

Chronic exudates and spring sap-ows

Tree exudates may be of two kinds, viz. slow but long-lasting (chronic slime-uxes) or vigorous but short-lived (spring sap-ows). Chronic exudates are caused by various factors,

such as mechanical injury or activity of invertebrate and microbial (especially bacterial) tree pathogens. Lasting for several months or even years, they harbour their own well-characterised microbial comunities. Many yeast species have been isolated and described from invertebrates associated with slime-uxes, such as wood-boring insects (see Phaff & Starmer 1987), ies (Phaff & Knapp 1956) and nematodes (e.g. Kerrigan et al. 2004). There is a high degree of host tree specicity which may be enforced by selection due to the nutrient composition of the sap or the presence of host defence compounds. Further, dispersal may be inuenced by the feeding preferences of insects which are the most important vectors for microbial consortium members (Phaff & Starmer 1987; Phaff 1990). In fact, yeasts play an important role in insect nutrition, and the insect gut harbours an immense diversity of yeasts, many of them belonging to as yet undescribed species (Suh et al. 2005). These yeasts may be spread by defaecation, as is well-known for Pichia spp. (Kurtzman 1998). In contrast to chronic slime-uxes, spring sap-ows are conned to the period of bud break when the high root pressure of many broad-leaved trees causes sap to rise. If the bark of trunks or branches is injured, massive bleeding

E-mail address: rwsweber@rhrk.uni-kl.de 0269-915X/$ see front matter 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycol.2006.09.015

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(sap-ow) may result. The xylem sap contains its highest concentrations of sugars and other nutrients in spring because these underground storage compounds are mobilised towards the expanding buds (Kallio & Ahtonen 1987; Ahtonen & Kallio 1989). Not surprisingly, therefore, the microbial composition of spring sap-ows differs from that found in chronic slime-uxes. Birch trees (Betula spp.) are particularly prolic producers of spring sap-ow, and their sap, being rich in monosaccharides (up to 1 % w/v), is collected commercially in Finland and North America for use as a health-promoting beverage or in the production of birch syrup as an alternative to maple syrup.

3. The diversity of yeasts suspended in birch sap

Although the natural occurrence of birch trees showing spring sap-ows is limited in time and space, characteristic microbial communities of yeasts, lamentous fungi and bacteria quickly establish themselves as biolms on solid surfaces, and as a planktonic phase of cells suspended in the sap itself. Golubev et al. (1977a,b), working in Betula pendula forests in central and northern regions of the European part of Russia, observed that a reproducible succession of yeast species developed as suspensions in spring sap-ow. Following an initial period of sterility, colonisation of the sap occurred in two phases. The rst phase, associated with rising daytime temperatures but continued night-time frosts, was characterised by the emergence of a wide diversity of yeast species, each at a low cell density. Frequently encountered species are listed in Fig. 1 under their current taxonomic names according to Barnett et al. (2000). Most of them belong to the Basidiomycota, the most abundant yeasts in this phase being Cryptococcus skinneri and C. magnus. Ascomycete yeasts were less obvious, examples including Nadsonia fulvescens var. elongata (Fig. 1) and members of the genera Metschnikowia, Candida and Aureobasidium (W.I. Golubev, personal communication). The occurrence of frost-free nights heralded the advent of a second phase in which the density of yeast cells suspended in the sap increased dramatically so that the sap became turbid. This coincided with the rise to dominance of two yeasts of low initial abundance, Trichosporon pullulans and Xanthophyllomyces dendrorhous (Fig. 1). Ludwig (1891) already noted a colour change of spring sap-ows from white to red. His red yeast (Rhodomyces dendrorhous) was probably X. dendrorhous, and the white one (Endomyces vernalis) may have been Trichosporon pullulans (see Kobayashi 1953).

(Italy), and on Carpinus betulus in the Upper Appennines (Italy). The most obvious overlaps with the Russian suspension consortia described by Golubev et al. (1977a,b) were the late-stage colonisers X. dendrorhous and T. pullulans at the species level, and Cryptococcus at the generic level (Fig. 1); another overlap was the red yeast Cystolobasidium capitatum (W.I. Golubev, personal communication). Nonetheless, the German and Italian biolms differed from the Russian suspensions in several important aspects, e.g. the prominence of the ascomycete yeast Hanseniaspora uvarum and the red basidiomycete yeast Cystolobasidium inrmominiatum, and the presence of mycelial fungi, notably Mucor hiemalis. These are fungi associated with plant remains and the soil, but repeated records of their presence in sap-ow biolms argue against chance contaminations.

5.

Interactions between consortium members

4.

The diversity of biolm-forming fungi

Fungi have been reported in the past as growing in biolms in spring sap-ows, but most of these older investigations are decient in details of species identication and/or give no indication of species composition of individual consortia (see Kobayashi 1953). For this reason, Weber and Davoli (2005) and Weber et al. (2006) examined spring sap-ow consortia on Betula pendula in Kaiserslautern (Germany) and Modena

By means of carefully executed single- and dual-culture laboratory experiments, Shkidchenko and Golubev (1980) found that T. pullulans and X. dendrorhous have faster growth rates than their main competitors, concluding that substrate competition, in addition to low freeze-thawing tolerance, is a likely explanation for the dominance of these two species in the nal phase of birch sap colonisation. Nutritional interactions have also been described. Thus, X. dendrorhous appears capable of utilising organic acids released by C. skinneri, resulting in the coexistence and enhanced growth of these two species in dual-culture in the absence of other competitors (Shkidchenko & Golubev 1980). A more intriguing case is that of infraspecic (i.e. strainspecic) competition in T. pullulans. Golubev et al. (2002) found that certain strains of T. pullulans produce a proteinaceous toxin capable of killing non-toxigenic strains of the same species from sap-ows but ineffective against other yeast species and even against T. pullulans strains from other habitats. Such killer toxins, called mycocins, are encoded by virus-like agents which bestow resistance upon the yeast cell containing them. Mycocins with an infraspecic action spectrum are common among ascomycete and basidiomycete yeasts (e.g. Saccharomyces; Magliani et al. 1997), whilst broader action spectra have been reported less frequently (Golubev 2006). In nature, killer strains are generally more abundant in nutrient-rich than nutrient-decient habitats (Starmer et al. 1987). Assuming that mycocin production poses an signicant metabolic burden with a retarding effect on growth rate, mathematical models can be developed to simulate the co-existence of killer and non-killer strains in transient and discrete habitats such ra n & Hoekstra 2003). In T. pullulans, the proas sap-ows (Cza portion of mycocin-producing cells among the total population in birch sap-ows rises steeply as the cell density of T. pullulans increases during spring, and mycocin production is lost again before the onset of the next growing season (Golubev et al. 2002). Other yeasts found in sap-ow consortia may also produce mycocins, e.g. C. inrmominiatum (Golubev et al. 2003) and H. uvarum (Schmitt et al. 1997), although their production in this habitat does not seem to have been examined as yet.

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R. W. S. Weber

Fig. 1 Summary of the presence of fungi in suspension (planktonic phase; Golubev et al. 1977a,b) and in biolms (Weber & Davoli 2005; Weber et al. 2006) of spring sap-ow consortia. Dominant species present in 50 % of subsamples are indicated in bold.

6. Survival and spread of consortium members

When the sap-ow dries up in late April or early May, the biolm remains as a dry plaque on the bark surface. Several of the yeasts forming sap-ow consortia, including the dominant X. dendrorhous and T. pullulans, have been isolated from such dried crusts one year after the event, and even from undamaged birch bark (Golubev et al. 1977b). It is therefore likely that these yeasts are essentially epiphytic. The apparent connement of X. dendrorhous to sap-ows of northern latitudes and its absence from the Modena consortium may be explained by its low temperature maximum for growth (2325  C; Golubev 1995). If the wound is sufciently low on the tree trunk or the sap drips downwards, fungi colonising it will reach the soil. In our experience, this is a common event (for images, see Weber et al. 2006). The soil is a reservoir for several fungi known to colonise sap-ows, such as lamentous species (Mucor

hiemalis, Fusarium spp.) and the yeasts Galactomyces geotrichum and Cryptococcus spp. Hanseniaspora uvarum (anamorph Kloeckera apiculata) is common on the surfaces of ripe and rotting fruits and has also been detected in the soil of fruit orchards (Phaff & Starmer 1987). Therefore, cells or spores of yeasts already present on the bark most probably represent the initial inoculum in early spring. Consortium members surviving in the soil may be disseminated by rainsplash, wind movements or insects. Further, if a sap-ow reaches the soil in early spring, it may provide a track for upwardly mobile lamentous fungi such as the fast-growing and sporulating Mucor and Fusarium spp. Insects are probably crucial as vectors for inoculum between different trees showing sap-ows. Fruit-ies and bees have been reported as carriers of several of the yeasts found in sap-ow consortia, an example being H. uvarum which is ingested by ies feeding on rotting fruits (Batra et al. 1973; Vacek et al. 1979; Phaff & Starmer 1987). Flies alight at sap-ows for feeding, and we have also noticed dung beetles (Geotrupes stercorarius) aggregating in high numbers at such

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sites. Efcient and rapid dispersal by insects may account for the spectacular phenomenon of every available stump in clear-felled plots being colonised by colouful biolms, as already noted by the earliest workers (Ludwig 1891).

Acknowledgement
I am grateful to Prof. W.I. Golubev and S. Herz for their constructive comments on early versions of this manuscript.

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