Compatible Solutes Tolerance of Water Stress and Translocation of Water

For fungi to grow under solute or matric stress, compatible solutes are needed to enable enzyme systems to function, and basidiomycetes are no exception in this respect (Magan, 1997). The key compatible solutes are the high-molecular weight sugar alcohols (polyols) mannitol and arabitol, and the low-molecular weight erythritol and glycerol. Glycerol and erythritol are outstanding in this respect and their accumulation is a major determinant of the water relations of xerotolerant and xerophilic micro-organisms (Brown, 1978; Magan, 1997). For desiccation tolerance, trehalose synthesis is particularly important. Potassium ions are easily accumulated by fungal cells and can serve as a compatible cytoplasmic osmoti-cum of low toxicity (Harris, 1981). However, at high concentrations, ionic solutes can become toxic (Luard, 1983). This was shown by studies in which stimulation of growth of some species occurred under slight osmotic stress (e.g. —0.5 to —1.5 MPa; Magan et al., 1995). This may be due to active accumulation of external KCl in the hyphae of the fungus (Brownell and Schneider, 1983), although less is known about K+ ion accumulation in mycelium of basidiomycetes than other fungi.

At equivalent concentrations, mannitol and arabitol are less effective as compatible solutes in reducing internal mycelial water potential when compared to erythritol and glycerol. However, as saprotrophic basidiomycetes are relatively sensitive the major polyols which are accumulated are indeed arabitol and mannitol. Many detailed studies on the cultivated mushroom A. bisporus have examined the effect of changes in solute stress on the relative accumulation of polyols in the mycelium, and subsequently in fruit bodies. This is because of the interest in understanding the way water and nutrients are translocated over long distances into fruit bodies and to optimise the production of the latter. Compatible solutes are accumulated to different extents depending on the external osmoticum (Table 1; Beecher et al., 2000). When glycerol is present in the medium it can readily enter the mycelium and improve the tolerance of water stress. This was accompanied by an increase in mannitol and trehalose. In ionic-modified media the accumulated solutes were erythritol and glucose. No trehalose was found in mycelia in this treatment.

It is also possible to examine the actual internal water potential (osmotic, matric and turgor pressure) of mycelia in relation to tolerance to water stress. This has been done by measuring the total water potential of the mycelial sample using a thermocouple psychrometer, then freezing the sample in liquid nitrogen, followed by thawing of the sample to release the ions which could then be measured (Beecher et al., 2000). By subtraction, the turgor potential can be determined. For A. bisporus, with both ionic water stress and matric stress, there

Table 1 Mean sugar and polyol concentrations (mM) in mycelia of A. bisporus colonies grown on media modified to different water potentials with glycerol and KCl

Treatments

Malt extract agar (-0.5 MPa)

Glycerol-modified (-2.5 MPa)

KCl-modified (-2.5 MPa)

Sugars

Trehalose

7.1

13.9

0

Glucose

1.1

0.5

3.1

Polyols

Arabitol

0.4

1.2

0.3

Mannitol

6.0

35.0

5.6

Erythritol

1.1

0.5

3.1

Glycerol

4.3

218.0

3.8

Note: Analyses carried out using HPLC with a RI detector. Source: From Magan et al. (1995).

Note: Analyses carried out using HPLC with a RI detector. Source: From Magan et al. (1995).

was an increase in turgor pressure and a significant decrease in osmotic and total water potential with increasing water-stress treatment (Fig. 4). This confirmed that matric stress induced the highest mean turgor at 25 °C. Thus, as greater water stress is imposed the internal water potential is changed by the synthesis of polyols, enabling the fungus to continue to function.

Interestingly, when polyol accumulation in fruit bodies of A. bisporus was examined, it appeared that in all the different tissues mannitol was by far the

Solutes Waters

Media water potentials (MPa) Figure 4 Effect of (a) ionic solute potential, (b) non-ionic solute potential and (c) matric water potential stress on relative total water potentials, osmotic potentials and turgor potentials of mycelium biomass of Agaricus bisporus measured using thermocouple psychrometry.

Source: From Beecher (2001).

Media water potentials (MPa) Figure 4 Effect of (a) ionic solute potential, (b) non-ionic solute potential and (c) matric water potential stress on relative total water potentials, osmotic potentials and turgor potentials of mycelium biomass of Agaricus bisporus measured using thermocouple psychrometry.

Source: From Beecher (2001).

Lo. Stipe Up. Stipe Gills (tin. Cap Peel The total concentrations of different sugar alcohols, glucose and trehalose in different tissue of first flush sporophores of the cultivated mushroom A. bisporus (Beecher, 2001). Key: Lo., lower; Up., upper; Inn., inner.

Figure 5

most important compound accumulated (Beecher et al., 2001). Fig. 5 shows an example of the concentrations of sugars and sugar alcohols in stipe, pileus and gill tissue in freshly harvested fruit bodies. This accumulation may contribute to a solute gradient which facilitates movement of water and nutrients from the mycelial cords or networks to the rapidly developing sporophores.

Since many saprotrophic basidiomycetes colonising wood produce mycelial cords and rhizomorphs they often move water and essential nutrients over long distances during fluctuating environmental conditions, especially availability of water. For example, Jennings and Watkinson (1982), working with S. lacrymans, suggested that mycelial cords facilitated the translocation of water and nutrients to developing fruit bodies (primordia). Work by Coggins et al. (1980) on droplet formation at hyphal tips of this species supported the suggestion of Jennings (1974) that translocation along hyphae and cords was due to a bulk flow of solution driven by hydrostatic pressure. This pressure was the result of soluble carbohydrates in the hyphae acting as osmotic solutes causing a flux of water into the hyphae as well as transporting solutes for growth to the hyphal tips. Indeed, Kalberer (1987) suggested that there was a water potential gradient from fruit bodies to mycelium in the compost and casing layer of A. bisporus which was responsible for the bulk flow of water.

Occurrence of endogenous adjustments of the concentrations of sugars and sugar alcohols in A. bisporus has been examined. Figs. 6 and 7 show the effect of colonies of A. bisporus growing from wet to dry conditions and dry to wet conditions respectively. The experiments were carried out in divided 9 cm Petri plates and KCl used to modify the water potential of one sector. The mycelia in each sector and the crossover zone were sampled for sugars and sugar alcohols, oo

Crossover

Crossover

13 2

Direction of growth

-►

(b) s x

Figure 6 The effect of changing water potential from freely available water to ionic waterstress conditions, in split agar plates, on (a) sugar alcohol and sugar alcohol accumulation in the mycelial biomass in three regions (wet — malt extract agar, MEA, crossover and ionic water stress, —2.48 MPa) and (b) effects on endogenous total water potential, osmotic and turgor potentials of the mycelial regions by A. bisporus grown on divided petri plates. Source: From Beecher (2001). The fungus was inoculated on the MEA agar side. Standard errors are within the size of the symbol.

Figure 6 The effect of changing water potential from freely available water to ionic waterstress conditions, in split agar plates, on (a) sugar alcohol and sugar alcohol accumulation in the mycelial biomass in three regions (wet — malt extract agar, MEA, crossover and ionic water stress, —2.48 MPa) and (b) effects on endogenous total water potential, osmotic and turgor potentials of the mycelial regions by A. bisporus grown on divided petri plates. Source: From Beecher (2001). The fungus was inoculated on the MEA agar side. Standard errors are within the size of the symbol.

and the internal water, turgor and osmotic potentials also measured using thermocouple psychrometry (Beecher, 2001). This showed that there were changes in endogenous synthesis of polyols, reflected by the actual changes in the mycelial turgor and water potentials in each condition and the crossover zone. These studies demonstrated that saprotrophic basidiomycetes can transport water and nutrients over long distances and continue to function under abiotic stress.

o Ph fe^l Trehalose IÎÎ3 Glucose Glycerol Erythritol Arabitol Mannitol

Crossover

Crossover

Direction of growth

Figure 7 The effect of changing water potential from ionic water stress (—2.48 MPa) to freely available water using an ionic solute on (a) sugar alcohol and sugar alcohol accumulation in the mycelial biomass of Agaricus bisporus in three regions (wet — MEA, crossover and ionic water stress, —2.48 MPa) and (b) effects on endogenous total water potential, osmotic and turgor potentials of the mycelial regions. The fungus was inoculated on the —2.48 MPa side. Standard errors are within the size of the symbol. Source: From Beecher (2001).

Figure 7 The effect of changing water potential from ionic water stress (—2.48 MPa) to freely available water using an ionic solute on (a) sugar alcohol and sugar alcohol accumulation in the mycelial biomass of Agaricus bisporus in three regions (wet — MEA, crossover and ionic water stress, —2.48 MPa) and (b) effects on endogenous total water potential, osmotic and turgor potentials of the mycelial regions. The fungus was inoculated on the —2.48 MPa side. Standard errors are within the size of the symbol. Source: From Beecher (2001).

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