It was long suspected that the very visible benefit given to plants by mycorrhizal infection was due to the supply of additional mineral nutrients. To prove this, it was necessary to show that the fungus could absorb mineral nutrients from the growth medium, transport them through its hyphae, and transfer them to the higher plant. The more general approach was also to prove that the mycorrhizal root system was more efficient than the non-mycorrhizal one. i.e. in the same soil it absorbed nutrients at a higher rate per unit amount of root.
Radioisotope techniques have been essential in establishing many of these facts. Absorption, translocation and transfer of mineral nutrients by the external hyphae of mycorrhiza were originally demonstrated in a series of papers by Melin and Nilsson (1950, 1952, 1953, 1955). The authors showed that isotopic phosphorus, nitrogen and calcium could be translocated through the hyphae of ectomycorrhizal fungi to their hosts. The basic system was simple, with the fungal hyphae being led over a barrier from a nutrient medium to the root system. An isotope was then added to the medium, and was subsequently detected in the host. The experiments showed the potential for uptake and transfer, but they were not quantitative and so could not be related to any specified level of improvement in plant nutrition.
The most direct measurements of the latter type were made by Pearson and Tinker (1975), and Cooper and Tinker (1978). In these studies, radioisotopes were supplied to vesicular-arbuscular mycorrhizal fungal hyphae which had grown over a barrier into agar, with the host plant growing in a soil-agar mix on the other side of the barrier. An important aspect of this system was that the entire shoot could be inserted into a whole-plant radioactivity counter. This allowed the non-destructive observation of the rate at which radioactivity appeared in the shoot. With these systems, it was shown firstly that 32P, 3 S and 65 Zn were taken up by vesicular-arbuscular mycorrhizal hyphae and transferred into the host. Additionally, the rates of uptake were measured, and it was shown that these were in the ratio P > S > Zn, as would be expected from the known composition of higher plants. By counting the hyphae crossing the barrier, and measuring their cross-section, estimates could be made of the minimum flux of these nutrients through the hyphae towards the plant. Finally, it was shown that the transfer rate was increased when the host plant was transpiring, which suggested a mass flow component within the fungal hypha, and that cytochalasin, a chemical which inhibits cytoplasmic streaming, brought the uptake to a halt.
Harley and co-workers, (Harley and McCready, 1950, 1952; Harley and Brierley, 1954), showed that excised beech mycorrhiza absorbed more 32P than did non-mycorrhizal roots. Under conditions of continuous supply of phosphorus, 90% of this phosphorus was retained in the fungal mantle. This was measured by excising the mantle from the "core" of root tissue using an ophthalmic scalpel. When the roots were placed in a phosphate-free medium, however, transfer rates from the sheath to the core increased. These basic conclusions with respect to transfer of phosphorus from the fungus to the plant were confirmed by Morrison (1957) using intact plants. He placed mycorrhizal and non-mycorrhizal pine seedlings, which had previously been fed with32P, into a phosphorus-free medium. He then monitored 32P levels in the shoots over 15 days, using a method similar to that of Pearson and Tinker (1975). He found that, while 32P levels in shoots of non-mycorrhizal plants remained constant, those of mycorrhizal plants increased steadily with time, indicating transfer of 32P from the roots. Thus, in a whole range of studies, radioisotopes have been essential in establishing that mycorrhizal hyphae can absorb mineral nutrients from the external medium and transfer them to the host plant.
The greater efficacy of vesicular-arbuscular mycorrhizal root systems in absorbing phosphorus from the soil was established by Sanders and Tinker (1971, 1973). They showed that inflow (uptake rate per unit length of root), was between three and four times higher for a mycorrhizal root, and that this inflow was much larger than could have arisen from the maximum rate of diffusion of phosphorus to the root surface, as shown by diffusion calculations. Radiotracers were not required in these initial studies, however they have been very useful in distinguishing between various interpretations of the results. By labelling the soil with 32P, it was possible to show that the specific activity of the phosphorus absorbed was equal to that of the soil solution, and similar in both mycorrhizal and non-mycorrhizal plants (Sanders and Tinker 1971). Uptake was therefore from the same isotopically-exchangeable pool (Larsen, 1967). A whole series of subsequent experiments have broadly confirmed the fact that both mycorrhizal and non-mycorrhizal plants obtain their phosphorus supplies from the same isotopically-labelled pool of soil phosphorus (Hayman and Mosse, 1972; Mosse et al., 1973; Powell, 1975; Pichot and Binh, 1976; Owusu-Bennoah and Wild, 1980; Gianinazzi-Pearson et al., 1981). It is important to under stand the precise implications of this technique. It is not able to detect shifts between the sorbed and solution phase phosphate which form the two components of the isotopically-exchangeable pool (Tinker, 1975), but it does show that phosphorus has not been "solubilized" from organic phosphates or from mineral phosphates that were not in isotopic equilibrium with the soil solution. If this had occurred, the specific activity of phosphorus in the plant would have been lower than that in the soil solution. Similar concepts can also be used to compare the efficiency of use of different fertilizer types by mycorrhizal and non-mycorrhizal plants.
Other types of experiments have used 32 P to support the theory that the increase in phosphorus uptake by mycorrhizal plants is due to absorption of phosphorus by the hyphae from beyond the depletion zones which develop around roots. In most soils, phosphorus is very poorly mobile. Thus any 32P applied is rapidly sorbed to soil solids and so can be used as a marker in experiments investigating the location of phosphorus uptake relative to the root. Using this technique, Hattingh et al. (1973) and Rhodes and Gerdemann (1975) clearly confirmed that external vesicular-arbuscular mycorrhizal mycelia can absorb phosphate up to 3-7 cm away from the root. In an ectomycorrhizal system, 32P applied to the cut end of a rhizomorph was transported 40 cm to the root (Finlay and Read, 1986b). In the latter case, autoradiography of the root system growing along a perspex plate was used to visualize transport.
These varied studies with radioisotopes have therefore given rise to the general conclusion that the mycorrhizal function is due to direct uptake by hyphae at a distance from the root, and not by some chemical modification of the soil. The results of Bolan et al. (1984) cannot be explained on this basis, but no other convincing explanation has been advanced for them. An experiment by Owusu-Bennoah and Wild (1979), which determined the depletion zone around roots by autoradiographic techniques with 32P, showed that mycorrhizal roots had a slightly wider depletion zone than a non-mycorrhizal root. This would not be expected if the additional inflow to the mycorrhizal root arises from a widely dispersed mycorrhizal network, with the host root continuing to function in the same way as before, as was assumed by Sanders and Tinker (1973) and Sanders et al. (1977). More information is obviously required on the distribution of external hyphae in soil. The general conclusion on mycorrhizal function therefore still stands, whilst emphasizing that there is further need for research on the general nutritional properties of mycorrhiza.
Due to its rapid immobilization in soil, 32P has been used to investigate the localization of phosphorus uptake relative to the distribution of the root systems of species competing for nutrients (Goodman and Collison, 1981; Caldwell et al., 1985; Perry et al., 1989). Perry et al. (1989) found that the competitive ability of two tree species to extract 32P from different soil depths was affected by the species and numbers of ectomycorrhizal fungi present on their root systems. Langlois and Fortin (1984), also using field-grown roots, found that pine mycorrhiza varied in their 32 P uptake capacity in a predictable way through the year.
One of the most interesting questions regarding mycorrhizal functioning in the field is the importance of hyphal connections between plants. Radioisotopes have been particularly useful in investigating transfer between plants. When two plants are grown in the same soil and 32P is applied to one plant, radioactivity is soon detected in the second plant (Chiariello et al., 1982; Whittingham and Read, 1982; Ritz and Newman, 1984). The amount of 32P transferred to the second plant is often higher when the plants are mycorrhizal and this has been used as evidence for mineral nutrient transfer via mycorrhizal connections (Whittingham and Read, 1982). There are, however, many problems in interpreting this type of result. For example, the presence of radioisotope in the receiver plant should not necessarily be interpreted as confirming net movement of the element concerned. It could merely be due to isotopic exchange between phosphorus pools in the two organisms (Newman, 1988). Alternatively, some exchange processes can be better explained as occurring through loss into the soil by the donor plant, and re-absorption by the recipient plant (Newman and Ritz. 1986). However, the results of other experiments indicate that phosphorus flow between the symbionts is only unidirectional, being from the fungus to the host (Finlay and Read, 1986b). Readers are referred to Newman (1988) for a full treatment of this topic.
Although research efforts have focused primarily on the uptake of phosphorus using 32P, other isotopes such as 45Ca, 35S, and Zn have been used as tracers for the uptake of the analogous ions by mycorrhizal plants (Morrison, 1962; Cooper and Tinker, 1978; Swaminathan and Verma, 1979; Ascon-Aguilar et al., 1986). The uptake of still other radioactive elements has been studied specifically because there can be raised levels of these elements in the environment as a result of nuclear activity. Thus vesicular-arbuscular mycorrhizal plants have been shown to absorb more 134Cs, 137Cs, 60Co and 90Sr than non-mycorrhizal plants (Jackson et al., 1973; McGraw et al., 1979; Rogers and Williams 1986). The results of these experiments must, however, be interpreted carefully. If the mycorrhizal plants are larger than the contrasted non-mycor-rhizal plants, an increase in the total uptake of any mineral element would not be surprising.
In plant nutrition studies, radioisotopes are often used to interpret the mechanisms of uptake. Inhibitors or low temperatures are applied to determine if, for example, the uptake (or in the case of mycorrhiza, the transfer) of the element requires an input of metabolic energy. This has been found to be the case for 32 P transfer between the fungal mantle and the root tissue in excised beech mycorrhizae (Harley and Brierley, 1955). Cooper and Tinker (1978) using 32P, 35S and 65Zn found suggestions of a lag phase in nutrient transfer between a vesicular-arbuscular mycorrhizal fungus and its host, an initial period during which uptake was extremely slow and which could not be explained by considerations of isotopic dilution along the root up into the shoot. It was therefore suggested that there was a need for the induction of some enzyme associated with uptake or transfer, but this matter was not settled. Jones et al. (1988) used 63Ni to show that nickel-tolerant mycorrhizal birch exhibited different patterns of metabolic and non-metabolic nickel uptake from those shown by non-mycorrhizal birch. These patterns suggested a mechanism for their differences in tolerance.
Rygiewicz and Bledsoe (1984), using 86Rb as a tracer for potassium, investigated why more potassium was retained in mycorrhizal than in non-mycorrhizal conifer roots. They performed a compartmental analysis and found that inward fluxes across the tonoplast were greater, and that effluxes out of the vacuole were smaller, in the fungal cells of the mycorrhiza than in root cells.
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