The isotope labelling methods described above also apply for the measurement of host-fungus carbon flow; however, as the mycobiont is an integral component of the root system it is impossible to distinguish 14C in root matter from 14C in intraradical fungal material; likewise the respiratory contribution of the individual symbiotic partners cannot be readily distinguished. The carbon flow to the fungus therefore has to be estimated on the basis of obtainable measurements in combination with some assumptions. Parameters which can be directly measured include intra- and extraradical fungal biomass, carbon incorporation by extraradical hyphae and loss of organic carbon from the roots. The respiration of the mycobiont and the carbon incorporation by its intraradical phase can be indirectly estimated by comparison of mycorrhizal and matched non-mycorrhizal plants, with the limitation that mycorrhiza may well affect the respiration of root cells. Methods for measurement of total respiration and possible methods for the direct measurement of hyphal respiration are discussed in Section II. C. 3.
It is possible to isolate external mycelium of arbuscular mycorrhizal fungi from soil by hand-picking with forceps (Sanders et al., 1977) or by wet-sieving (Jakobsen and Rosendahl, 1990b), but it is difficult to get rid of all fragments of soil organic matter. The peat growth substrates used in ectomycorrhizal research amplify this problem. Consequently gravimetric methods are of limited use for quantifying the biomass of internal fungal components of the roots. The biomass of hyphae is related to their biovolume, which may be calculated from microscope measurements of length and diameter of the hyphae. Kucey and Paul (1982b) measured the length of intraradical hyphae after high-speed blending of colonized roots. Methods for the measurement of external hyphae are discussed elsewhere (Methods in Microbiology, Vol. 24, Chapter 3). It is desirable to group the hyphae in classes according to their diameter as the use of an overall mean diameter may greatly underestimate the true biovolume (Bááth and Sóderstróm, 1979; Schnürer et al., 1985). Biovolume may be converted to biomass on the basis of density and dry matter content. Conversion factors
(g dry wtcm"3) may be obtained from pure cultures of fungi (Van Veen and Paul, 1979; Bakken and Olsen, 1983) or from hyphae picked from their natural substrates (Lodge, 1987). The conversion factors for hyphae from leaf litter were 0.19-0.23 (Lodge, 1987) while an average of 0.23 was obtained for 10 different fungi (Bakken and Olsen, 1983).
Fungal biomass may also be determined from chemical measurement of compounds which are specific to fungal tissue (Whipps et al., 1982). Chitin, a polymer of /V-acetylglucosamine and the major wall component in all mycorrhizal fungi, may be quantified colorimetrically after alkaline (Hepper, 1977; Bethlenfalvay et al., 1981) or acid (Vignon et al., 1986) hydrolysis. The analytical procedure involving acid hydrolysis is faster than the alkaline hydrolysis, but has the disadvantage that it is not strictly specific as aldehydes derived from plant material are also sensitive to the colour reaction (Vignon et al., 1986). Glucosamine contents in roots may be converted to fungal biomass using the specific glucosamine content of clean external mycelium. This conversion factor ranged from 21-40 ¡xg glucosamine mg"1 fungal dry weight in five fungi forming arbuscular mycorrhiza (Hepper, 1977; Bethlenfalvay et al., 1982), indicating that conversion factors should be determined in each experimental situation. The use of conversion factors obtained from pure cultures of fungi forming ericoid and ectomycorrhiza should be avoided until it has been shown that the conversion factors are unaffected by changed environmental conditions. The use of these conversion factors further necessitates the assumption that the specific glucosamine content is similar for intra- and extraradical hyphae. The chitin method is suited for determination of biomass of mycorrhizal fungi in pathogen-free roots, while its use on external hyphae is hampered by the native amino sugar component (Parsons, 1981) and chitin-containing non-mycorrhizal organisms in soil. Pacovsky and Bethlenfalvay (1982) used a rather complicated wet-sieving procedure to get rid of the amino sugar component and obtained reasonably large differences between chitin content in soil from mycorrhizal and non-mycorrhizal plants. In general, the method is suitable only for soils low in organic matter and a considerable degree of variability may be introduced due to the many steps in the procedure (Bethlenfalvay and Ames, 1987).
The chitin assay measures total chitin in both viable and dead hyphae. Methods for measuring the biomass of viable hyphae are important in relation to functional aspects of the mycorrhiza. Living biomass may be determined microscopically from total biovolume and the proportion of biovolume showing metabolic activity after staining with fluorescein diacetate (Schubert et al., 1987) or iodotetrazolium (Sylvia, 1988). Recently, image-analysis techniques have been applied to quantify the activity of arbuscules measured as the intensity of staining for succinate dehydrogenase with nitroblue tetrazolium (S.E. Smith, pers. commun.).
The availability of biochemical assays for detecting compounds which are specific to living mycorrhizal hyphae would facilitate a more exact measurement of viable biomass of mycorrhizal fungi. Ergosterol is the dominant sterol in most fungi (Weete, 1974) and is becoming increasingly popular as an index of fungal biomass in soil (Grant and West, 1986) and in plant material (Newell et al., 1988). Salamanowicz and Nylund (1988) measured the ergosterol content of hyphae of ectomycor-rhizal fungi by HPLC and found only small differences due to fungal species and time; values ranged from 2.88-4.03 mg ergosterol g_1 dry matter. Ergosterol analysis may be performed on samples as small as 2 mg fresh wt and was therefore superior to the chitin method for quantifying fungal growth during early stages of ectomycorrhiza formation (Martin et al., 1990). Ergosterol analysis is not adequate for measuring biomass of external hyphae of ectomycorrhizal fungi in unsterile substrates as the results would be influenced by the ergosterol content of the background fungi. Ergosterol has also been identified in arbuscular mycorrhizal fungi but in general concentrations seem to be very low (Beilby, 1980; Beilby and Kidby, 1980a; Nagy et al., 1980; Nordby et al., 1981). The arbuscular mycorrhizal fungi contain large quantities of a number of fatty acids which are not found in plants (see Section II.C.l). When quantification is achieved these fatty acids, especially the 16:l(llc) and 18:3(6c,9c,12c) may turn out to be useful indicators of the living biomass of arbuscular mycorrhizal fungi. The composition of fatty acids differs between fungal taxonomic groups (Weete, 1974). In Ascomycetes and Basidiomycetes the 18:3 component has a (9c, 12c, 15c) conformation in contrast to the (6c,9c, 12c) found in Phycomycetes. Furthermore, the fatty acids of Zygomycetes differ in their degree of unsaturation from that of Chytridomycetes and Oo-mycetes, as the two latter groups have a higher potential for long-chain polyunsaturated (> 18:3) fatty-acid synthesis than the Zygomycetes. There is a need for more detailed comparisons of the fatty acids in arbuscular mycorrhizal fungi and in saprotrophs isolated from soil, in order to assess the potential value of the fatty acids as markers for external mycorrhizal hyphae.
The carbon flow to the external hyphae is of importance not only for the study of the carbon balance of the symbiosis but also to the distribution and cycling of carbon in the soil ecosystem. This subject has been reviewed recently by Finlay and Sóderstróm (1991). The use of 14C labelling has made it possible to quantify the amount of carbon which is incorporated by the hyphae.
Methods used for the sampling of hyphae for scintillation measurements are not always well described. Bevege et al. (1975) quantified the 14 C activity in the total amount of external hyphae from pulse-labelled Trifolium subterraneum L. and Kucey and Paul (1982a) picked hyphae for 14C determination from soil cores taken from pots with Vicea faba L. but no details on sampling techniques were provided. Visible hyphal strands were picked with forceps from a sandy soil of a 27 cm x 27 cm x 2 cm Plexiglass chamber with Pinus ponderosa Laws, and specific activity determined (Norton et al., 1990). In a carbon translocation study with mycorrhizal Pinus sylvestris L. grown in peat in a similar Plexiglass chamber the peat with hyphae was dissected in squares of equal size and the 14C activity determined for each square (S. Erland, pers. commun.). A quantitative sampling of external hyphae is facilitated from growth systems including a special hyphal compartment, where roots are prevented from crossing the barrier between the hyphal compartment and the root compartment. The hyphal compartments of the systems developed by Rygiewicz et al. (1988) (see Section II.C.3) contained glass fibre filter paper moistened with nutrient solution. At harvest hyphal dry weights were determined gravimetrically and their 14C content measured (Miller et al., 1989; Andersen and Rygiewicz, 1991). This system is designed also to allow for measuring 14C02 development in the hyphal compartments and consequently the carbon flow to the hyphae and their growth efficiency may be determined.
The following method is suitable for the quantification of carbon incorporation by external hyphae of arbuscular mycorrhiza (Jakobsen and Rosendahl, 1990b). A soil container is divided into two compartments by a nylon or stainless steel mesh which prevents root growth but allows hyphae to penetrate. A 25-35 ¡xm mesh will eliminate root penetration, even of fine-rooted grasses. The compartmentation may take different forms depending on the container used. A simple solution is to grow the roots in a mesh bag inserted in a pot or PVC tube filled with soil (Fig. 4). The PVC tubes may be hermetically sealed for 14C02 labelling very easily by using standard PVC closing sockets (Fig. 4) in combination with O rings and Terostat. At harvest the external mycelium may be quantitatively extracted by repeated wet-sieving and decanting from either the whole soil volume or from a subsample of the soil. Hyphae are collected on a fine sieve (40-50 ;u,m mesh). At least 5-10 resuspensions of the soil in water followed by decanting are needed. Care must be taken to collect hyphal aggregates trapped in the
So. mm PVC TuOe
100 t/T mesh H>MOe< band
100 t/T mesh H>MOe< band
Gap Hx plant stem
Gap Hx plant stem
Standard PVC closing sockets MW copper hiOing
Standard PVC closing sockets MW copper hiOing
Fig. 4. An example of a growth system suitable for measuring incorporation of 14C by the external hyphae of arbuscular mycorrhiza. Hyphae can grow out into the hyphal compartment surrounding the mesh bag containing the roots. The two closing sockets are mounted before labelling.
sand precipitate in the suspension beaker; a magnifying glass may be useful in this respect. The method is probably not adequate for soils high in organic matter. The hyphae on the sieve are washed and analysed and their 14C content expressed on the basis of dry weight obtained gravimetrically or by conversion from biovolume (Bakken and Olsen, 1983). The radioactivity in the total amount of external hyphae may be calculated if the hyphal density is assumed to be similar inside and outside the root compartment. This will depend on the size of the hyphal compartment and on the mycorrhizal fungus used, but in any case the hyphal density is never smaller in the root compartment than in the hyphal compartment (I. Jakobsen, unpubl. res.). Another problem is that it is not yet known whether similar amounts of hyphae inside and outside the root bag are likely to incorporate equal amounts of 14C. Adaptation of the system with a hermetically sealed hyphal compartment (Andersen and Rygiewicz, 1991) to arbuscular mycorrhiza would allow for respiration measurements and the direct measurement of carbon flow to the hyphae.
3. Extra-mycorrhizal loss of organic carbon from roots
In addition to respired C02 and carbon contained in external mycor-rhizal hyphae, roots also lose considerable amounts of carbon as exudates (carbohydrates, organic acids, amino acids), secretions (polymeric carbohydrates) and lysates (sloughed cells or part of cells) (see Whipps, 1990). This non-hyphal flow of organic carbon may be influenced by mycorrhiza (Graham et al., 1981) and should be quantified in studies of carbon balance in mycorrhiza. However, exudates are rapidly metabolized by micro-organisms in the growth medium (Minchin and McNaughton, 1984) and meaningful data can therefore be obtained only from aseptically grown plants (Laheurte and Berthelin, 1986; Lipton et al., 1987). This problem was addressed in studies of arbuscular mycorrhiza by removing roots from soil, washing and treating roots with antibiotics and subsequently collecting exudates in CaCl2 for a number of hours (Graham et al., 1981; McCool and Menge, 1983; Schwab et al., 1983). However, it is likely that the mechanical disturbance of roots and the removal of micro-organisms would have affected exudation patterns (Whipps, 1990). Quantification of carbon loss from undisturbed roots of soil-grown plants is best performed by means of 14C02 pulse-labelling of shoots. Carbon-14 in soil samples including external mycelium of arbuscular mycorrhiza was measured by Snellgrove et al. (1982), Harris et al. (1985) and Douds et al. (1988). Before analysis of 14C, the external hyphae may be quantitatively removed (see Section III.C.2). After filtration of the washing water to remove all clay, silt and sand fractions, soluble 14C compounds are measured in subsamples from the filtrate while insoluble 14 C is measured in subsamples from the material retained by the filter (Whipps and Lynch, 1983).
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