THE SIGNIFICANCE OF MYCORRHIZAL NODULES OF AGATHIS AUSTRALIS

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1 New Phytol. (1967) 66, THE SIGNIFICANCE OF MYCORRHIZAL NODULES OF AGATHIS AUSTRALIS BY T. M. MORRISON AND D. A. ENGLISH Lincoln College, Canterhurv, Nezv Zealand {Received 18 October 1966) SUMMARV Experiments using excised nodules of kauri showed that vesicular-arbuscular mycorrhizal infection markedly stimulated phosphate absorption by these organs. Small but significant fixation of nitrogen was also noted in excised nodules, whether mycorrhizal or not. INTRODUCTION Baylis, McNabb and Morrison (1963) reported that growth of mycorrhizal plants of Podocarpus totara and P. dacrydioides was greater than that of non-mycorrhizal plants in an infertile soil. Evidence of growth stimulation in plants carrying Rhizophagus or Endogone mycorrhizas is accumulating (Baylis, 1959; Mosse, 1963; Gerdeman, 1964; Daft and Nicholson, 1966; Holevas, 1966) and some evidence that this stimulation is due to enhanced phosphate absorption by these mycorrhizas compared with non-mycorrhizal roots has been presented in these reports. No such experiments have been reported for Podocarps and the stimulatory effect of these mycorrhizas remains unexplained. Kauri {Agathis australis) Araucariaceae in common with the Podocarps in New- Zealand has all but a few of its short roots converted to nodular structures. The nodules are infected with a typical vesicular-arbuscular fungal symbiont of the type regarded by Mosse as Endogone. In addition to this endophyte other cortical cells of the nodules are often invaded by a second endophyte which resembles an actinomycete in structure. Details of these infections will be published elsewhere. Bergerson and Costin (1964) have shown that nodules of Podocarpus lawrencei are capable of fixing some atmospheric nitrogen and have suggested that this would account for the pioneering ability of this species. An ability to extract phosphate from poor soils would also aid pioneer species. Agathis australis (Kauri), however, is a climax tree in northern New Zealand and is ecologically distinct from the scree-dwelling Podocarpus species used by Bergerson and Costin. Harley and his co-workers (Harley, 1959) and Morrison (1954, 1962) have demonstrated that ectotrophic mycorrhizas of beech and pine respectively show similar enhanced absorption of phosphate from nutrient solutions and soil compared with non-mycorrhizal roots and have concluded that this may be a major advantage to the higher plant of mycorrhizal infection. The experiments reported here were planned to find if this conclusion applied to endotrophic mycorrhizas of kauri and to test the ability of these structures to fix atmospheric nitrogen. 245

2 246 T. M. MORRISON AND D. A. ENGLISH METHODS Mycorrhizal infection of seedlings was obtained by growing plants for a period in soil taken from a stand of mature trees, and non-mycorrhizal plants by growing them on a similar soil to which a nutrient solution lacking phosphate had been added. In this way rhizosphere differences between the two major treatments might be less than when using unsterilized and sterilized soils. Microscopic examination showed that in plants herein called mycorrhizal at least 50" of foot or nodule cortical cells were infected while in non-mycorrhizal plants not more than 10 were infected with the typical endophyte. All plants became nodulated and 3 weeks prior to the experiments they were transferred to a nutrient solution lacking only phosphate so that they would be of similar nutrient status at the time of the experiments. All plants were of the same age and appeared to be of similar \ igour. Short segments of roots w ith attached nodules were excised shortly before treatments began. Nodules were then cut quickly from the segments and placed in a solution of labelled phosphate containing sufficient H3^^PO4 to give an activity of 2^ or 50 ^Cijl and Na2PO4, 2H2O at 0,3 mm concentration (ph 5,0), The solution in conicalflaskswas shaken in a water bath maintained at the required temperature. After this exposure to ^^P the roots were filtered out and washed several times with water to remove surface radioactivity, dried, weighed and assayed for radioactivity by standard procedures. Samples were subdivided into sub-samples containing approximately 2 mg of material. Results recorded for each treatment arc thus averages of not less than three to five sub-samples and are expressed as counts/min/mg of dried material. RESULTS Mycorrhizal and non-mycorrhizal nodules w'ere immersed in phosphate solution for up to 2 hours at 25" C, Estimation of phosphate absorption by the nodules after 5, 10, 30, Fig, I, Specific activity of mycorrhizal ( ) and non-mycorrhizal ( ) nodules immersed in NaHj^^PO^ solution at 25 C, 60 and 120 minutes are shown in Fig. i. A clear enhancement of phosphate absorption was demonstrated by mycorrhizal nodules over the whole 2-hour period.

3 Mycorrhizal nodules of Agathis E Fig. 2. Specific activity of nodules immersed in NaH2^-PO4 solution, (a) Mycorrhizal, (b) non-mycorrhizal., 25' C;, 19 C; T, 13 C; :, i^ C. 600 r Minutes Fig. 3. Specific activity of mycorrhizal (circles) and non-mycorrhizal (triangles) roots after immersion in NaH^^^FOi. Open symbols, 1 C; solid symbols, 25'" C.

4 248 T. M. MORRISON AND D. A. ENGLISH In order to make some distinction between physical and metabolic uptake, mycorrhizal and non-mycorrbizal nodules were immersed in phosphate solution held at temperatures of I, 13, 19 and 25 C for periods of up to i hour. Estimation of pbospbate absorption by tbe nodules after 5, 10 and 60 minutes are shown in Fig. 2. Lowering tbe temperature affected absorption even after only 5 minutes and therefore metabolically dependent pbospbate uptake was a significant part of absorption in this short time in botb mycorrbizal and non-mycorrbizal nodules. At all temperatures the presence of the endophyte enhanced absorption of phosphate by nodules. Occasionally non-nodulated short roots are found on mycorrbizal and non-mycorrhizal plants. Samples of these were immersed in phosphate solutions held at temperatures of Stoppei Latex rubber 5eal Capillary Fig. 4. Container for exposure of exised nodules to '""^Xi. i" C or 2s' C for periods of up to i hour. Estimation of phosphate absorption by the roots after s, 10 and 60 minutes are shown in Fig. 3. As in the case of nodules, mycorrbizal infection enhanced phosphate uptake, but here the stimulation was less marked. In the first 10 minutes absorption was similar in mycorrhizal and non-mycorrhizal roots at both 25 C and 1 C whereas in nodules a doubling of phosphate absorption was associated with mycorrhizal infection even after 1^ minutes. Nodules were exposed to an atmosphere enriched with ^^Nj. Plant material was obtained from tbree sources: (a) heavily nodulated mycorrhizal roots from a mature tree, (b) mycorrbizal nodules from infected seedlings obtained as described above under 'Methods', and (c) non-mycorrhizal nodules from uninfected seedlings obtained as described above under 'Methods'. Approximately i g fresh weight of nodules was placed in a container of 30 ml capacity which was then filled completely with water. Gas was introduced in measured volumes via the rubber seal with a gas syringe, water being displaced through the capillary at the

5 Mycorrhizal nodules of Agathis 249 base of the container. The final atmosphere consisted of: 9.5% '^^N,, 0.5",, ''^N,, 20",^ O, and 70% argon. The flask and contents were then submerged in a water bath kept at 25' C for 24 hours. Soluble nitrogen was extracted from nodules in 3 N-HCl, estimated by normal Kjeldahl digestion and assayed for '-"^N. Results of this test for nitrogen fixation are given in Table i. Table i. Atoms enrichment in soluble nitrogen from nodules exposed to an enriched ' '^V atmosphere (a) Mycorrhizal nodules from mature tree Sample i Sample 2 (b) Mycorrhizal nodules from seedlings (c) Xon-niycorrhizal nodules from seedlings O.OIO O.OII ±0.001 ±0.002 ±0.001 ±0.001 DISCUSSION The results presented here explain the reports of higher phosphate content of mycorrhizal plants, infected endotropically presumably with Endogone, as compared with nonmycorrhizal plants. The greater phosphate uptake of mycorrhizal nodules is very striking. Indeed the course of absorption and the magnitude of the differences which have been observed is somewhat similar to those of infected and uninfected beech and pine root systems (Harley, 1959; Morrison, 1962). In the latter the fungus is predominantly outside the host and phosphate accumulation is into the fungal tissue. In the endotrophic mycorrhiza here considered, both host tissue and its enclosed fungal hyphae may be able to absorb phosphate directly and it seems likely that both components may contribute to the absorption rate observed. However, not only is the fungus digested by the host cells but also metabolic release of phosphate to the host by intact hyphae is possible. Hence even if the primary accumulation is into the fungus, the phosphate absorbed is likely to be available to the whole host and fungus system. The effect of temperature on the rate of absorption is such as would be expected if the process were metabolically dependent. The two phases of the uptake curve an initial rapid, followed by a slower phase may suggest two processes of absorption rapid absorption into the fungus coupled with a slower movement from the fungal hyphae to the host. However, the role of host cytoplasmic membranes in the movement of ions into the enclosed fungal hyphae is not clear from these experiments. It is possible that a study of the kinetics of the process, perhaps on the lines of the work on ectotrophic mycorrhizas (Harley and Loughman, 1963; Jennings, 1964) might elucidate the precise relationships between host and fungal tissues. The evidence for nitrogen fixation in kauri nodules is not clear. Obviously some enrichment with '^N occurred but it was inconsistent. Exposure of Coriaria and Discaria nodules (see Morrison, 1961) to similarly enriched atmosphere at the same time as the kauri nodules resulted in enrichments of up to ten times that of kauri nodules. Values for similar field-grown samples were widely different and we have found that this variation in nitrogen fixation also occurs in Podocarp nodules. This inconsistent and low fixation could be due to the presence of some transient microbial member of the rhizosphere which is able tofix atmospheric nitrogen but this would not account for an enrichment of atoms % recorded here or the 2.6 atoms % obtained in this laboratory with nodules from Podocarpus alpinus. The inconsistency is more likely to be due to

6 2SO T. M. MORRISON AND D. A. ENGLISH some otber factor such as a seasonal or ageing effect. The low rate of nitrogen fixation occurring in kauri nodules, which is consistent with the ecological status of this plant, could be carried out by the Actinomycete-like organism in the cortical cells. We can be fairly sure that the presence of a mycorrbizal fungus did not account for it since some fixation occurred as well in non-mycorrbizal nodules. ACKNOWLEDGMENTS We are indebted to the New Zealand Forest Service, Lottery Distribution Committee and University Grants Committee for research funds. REFERENCES BAYLIS, G. T. S. (1959). Effect of vesicular-arbuscular mycorrhizas on growth of Griselinia littoralis (Cornaceae). Nezo PhvtoL, 58, 274. B.WLis, G. T. S., McN.\BB, R. F. R. & MORRISON, T. M. (1963). The mycorrhizal nodules of Podocarps. Trans. Br. mvcol. Soc, 46, 378. BERGERSON, F. J.' & CosTiN, A. B. (1964). Root nodules on Podocarpus lau-rencei and their ecological significance. Aitst. J. biol. Sci., 7, 252. DAFT, M. J. & NICHOLSON, T. H. (1966). Effect of Endogene mycorrhiza on plant growth. New Phytol., 65, GERDEM.-^N, J. W. (1964). The effect of mycorrhiza on the growth of maize. Mycologia, 56, 342. HARLEY, J. L. (19S9). The Biology of Mycorrhiza. London. HARLEY, J. L. & LOUGHMAN, B. C. (1963). The uptake of phosphate by excised mycorrhizal roots of the beech. IX. The nature of the phosphate compounds passing into the host. New Phytol., 62, 350. HoLEVAS, C. D. (1966). The effect of a vesicular-arbuscular mycorrhiza on the uptake of soil phosphorus by strawberry (Fragaria sp. var. Cambridge Favourite). J. hort. Sci., 41, 57. JENNINGS, D. H. (1964). Changes in the size of the orthophosphate pools in mycorrhizal roots of beech with reference to absorption of the ion from the external medium. Nezo Phytol., 63, i8i. MORRISON, T. M. (1954). Uptake of phosphorus-32 by mycorrhizal plants. Nature, Loud., 174, 606. MORRISON, T. M. (1961). Fixation of nitrogen.. 15 by excised nodules oi Discaria toumatou. Nature, Lond., 189, 94s. MORRISON, TT. M. (1962). Absorption of phosphorus from soils by mycorrhizal plants. Nezu Phytol., 61, 10. MossE, B. (1963). Vesicular-arbuscular mycorrhiza: an extreme from of fungal adaptation. Symp. Soc. gen. Microbiol., 13, 146.

EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH

New Phytol. (1969) 68, 945-952. EFFECT OF ENDOGONE MYCORRHIZA ON PLANT GROWTH II. INFLUENCE OF SOLUBLE PHOSPHATE ON ENDOPHYTE AND HOST IN MAIZE BY M. J. DAFT AND T. H. NICOLSON Department of Biological