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The effect of photon irradiance on phosphate uptake and growth of onion plants was investigated in an experiment aimed at determining whether development and activity of mycorrhizal infection and arbuscules are linked to physiological effects. Mycorrhizal (inoculated with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe) and non- mycorrhizal plants of Allium cepa. L. were grown at three soil phosphate levels and two photon irradiances (410 and 190 mol m-2 s-1). There was one harvest at 6 weeks. Data were obtained for phosphate uptake (specific uptake rate), plant growth and mycorrhizal infection (total and active infection by trypan blue staining and succinate dehydrogenase (SDH) activity respectively). There was a positive growth response to mycorrhizal infection at the lower P levels (P0 and P1) but not at P2 with high irradiance. This response was smaller at low irradiance. Furthermore, data for root/shoot and fresh-weight/dry-weight ratios indicated that allocation of dry matter was altered in mycorrhizal plants in accord with their increased demand for photosynthate. Reduced irradiance was associated with lower rates of P uptake in all treatments, with the greatest effect in mycorrhizal plants at P0 and P1. These reductions were not associated with reductions in incidence or intensity of mycorrhizal infection nor in the development of arbuscules. At P2 the incidence of infection was reduced under low irradiance, but development of arbuscules within infected regions of the roots remained the same as in other treatments. The use of nitroblue tetrazolium chloride as a vital stain for SDH activity showed that during the 6-week period of the experiment, all the infection revealed by trypan blue staining was physiologically active. Branched arbuscules (counted in freehand sections of fresh roots) always had SDH and alkaline phosphatase activity. Thus, in young plants, observations of arbuscules gives a good indication of the amount of active infection. This may not be so in older roots.

Many studies of interactions between the organisms which form biotrophic symbioses have concentrated on extracellular events such as signals and responses that involve modifications to cell wall metabolism. It is clear, however, that formation of a biotrophic association must also involve signals and responses at the membrane level and modifications to membrane activity resulting in changes in the transfer of nutrients. In parasitic biotrophs the modifications to membrane transport result in unidirectional transport (at least in the long term). In mycorrhizal associations, lichens and N2-fixing symbioses, it appears that well regulated bidirectional transport of nutrients between symbionts must occur, thus allowing a persistent compatible (and mutualistic) symbiosis. Evidence for such membrane modifications comes from changes in ATPase activity. The overall question to be considered in mutualistic symbioses is whether nutrient transfer processes have analogies elsewhere in the physiology of plants or whether 'new' transport events are switched on as a result of interactions between the organisms.

Methods for the objective measurement of vesicular-arbuscular mycorrhizal infection in the roots of four species of host plant are described. The methods are based on vital staining of transverse sections of fresh roots, followed by evaluation of occurrence and/or numbers of living arbuscules and hyphae per section. The most useful method involved freeze-sectioning roots embedded in gelatin containing glycerol and DMSO, followed by staining the sections with nitroblue tetrazolium overnight to reveal succinate dehydrogenese activity. Two systems of computer-aided image analysis were evaluated for measurement of the number, perimeter and cross-sectional areas of intercellular hyphae and arbuscules. This approach, which is clearly objective, is an advance on previous methods which were based on ranking sections or segments of root with respect to the intensity of development of arbuscules. The data were used to calculate the area of interface between plant and fungus in infected roots. Results are presented showing that the contribution of arbuscules to the total infection and area of interface declines markedly as plants and infections age. Differences in infection in different host/fungus combinations are also described.

Three experiments are described. Rapid establishment of vesicular-arbuscular mycorrhizas in roots of T. subterraneum cv. Mt Barker, using natural soil inoculum, was associated with improved nodulation, increased nitrogenase activity per plant (nmol C2H2 reduced per plant per hour) and increased nodule efficiency on the basis of nodule volume (nmol C2H2 reduced per mm nodule per hour). In two experiments (on soil low in nutrients), this increase occurred before any positive growth response to mycorrhizal infection was apparent. In all experiments, mycorrhizal roots had a higher phosphorus concentration (%P) than did non-mycorrhizal roots. This difference, which was evident before any differences in total plant phosphorus were detected, was not accompanied by an increase in nodule phosphorus concentration, so that differences in nodule efficiency could not be attributed to differences in this parameter. In the third experiment (on soil with higher nutrient levels), establishment of mycorrhizas was also accompanied by increased growth, phosphorus and nitrogen contents within a 35-day experimental period. Phosphorus inflow into roots (moles P per cm root per second) was higher in mycorrhizal plants. Delay in formation of mycorrhizas (by reduction in the amount of inoculum in soil) was accompanied by lower inflow, and delay in both the establishment of high root phosphorus concentration and in the onset of enhanced nodulation and nitrogenase activity.

Nodulated Medicago sativa cv. Europe plants, both non-mycorrhizal and mycorrhizal (inoculated with Glomus mosseae), were grown in sand or soil with a range of phosphate levels. The following parameters were measured: intensity of mycorrhizal infection, intensity of nodulation, growth, phosphate content, nitrogenase activity (acetylene reduction) and nitrogen content. Both nodulation and mycorrhizal infection had occurred within 2 weeks of inoculation with the appropriate endophytes. Major increases in dry weight of mycorrhizal plants were not apparent until approximately 10 weeks from inoculation. However, mycorrhizal plants showed more extensive nodulation, coupled with higher rates of nitrogenase activity from 2 weeks onwards, and at the final (12-week) harvest had higher values of % N. Phosphate content of mycorrhizal plants (g P/g dry wt) was also greater than non-mycorrhizal plants at 7 weeks. By 10 or 12 weeks, when significant mycorrhizal enhancement of growth was apparent, the total nitrogen and total phosphorus per mycorrhizal plant were also higher, but nitrogenase activity and phosphate content measured on a dry weight basis showed no significant differences between mycorrhizal and non-mycorrhizal plants. It is clear that mycorrhizal enhancement of phosphate uptake and nitrogen fixation precedes any effect on growth and the results indicate the importance of the time factor in the development of this tripartite symbiosis between legume, Rhizobium and mycorrhizal fungus.