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Nitrogen fixation by nodulated species of Pavetta and Psychotria
Margaretha Mes Institute for Plant Physiology and Biochemistry, University of Pretoria, Republic of South Africa
Nitrogen Fixation by Nodulated Species of Pavetta and Psychotria N. GROBBELAAR and E. G. GROENEWALD With 1 figure Received January 9, 1974
Summary Leaves bearing bacteria-filled nodules from seven Pavetta and two Psychotria species were tested for nitrogenase activity by the acetylene reduction method for up to 24 hours with negative results. Four of the Pavetta species and the two Psychotria species used in the acetylene reduction tests were also grown in a nitrogen-free culture solution for 75 days. The plants developed severe symptoms of nitrogen deficiency and no increase in their nitrogen content could be detected during the experimental period.
Key words: Leaf nodules; nitrogen fixation; Pavetta; Psychotria.
Introduction
Bacterial leaf nodules are a common feature of many species of Pavetta and Psychotria (BREMEKAMP, 1933, 1934). There is considerable uncertainty about the nitrogen fixing ability of such plants. None of the reports (VON FABER, 1912, 1914; SILVER et aI., 1963; GROBBELAAR et aI., 1971) which mentions positive results for nitrogen fixation is convincing. In some cases (VON FABER, 1912, 1914; RAO, 1923; SILVER et aI., 1963; BETTELHEIM et aI., 1968) it is claimed that the endophyte was isolated and shown to be able to fix nitrogen. However, in no case was it unequivocally established that the isolate was the true endophyte of the leaf nodules. To add to the uncertainty, BOND (1958), BECKING (1971) and SILVESTER and ASTRIDGE (1971), using sensitive modern methods, were unable to demonstrate nitrogen fixation in nodulated species for which other workers reported positive results. HUMM (1944) also did not find evidence for nitrogen fixation in experiments with Psycho tria punctata. In view of the above, we have now more rigorously investigated a wider range of nodulated species for their ability to fix nitrogen. We were prompted to undertake the present study because in our initial investigation (GROBBELAAR et aI., 1971) a) The UN-method, of which we have had considerable experience, gave negative results when applied to nodulated leaves as well as whole seedlings of Pavetta assimilis ander various conditions.
z.
Pflanzenphysiol. Bd. 73. S. 103-108. 1974.
104
N. GROBBELAAR and E. G. GROENEWALD
b) The small increase (7 mg) in the nitrogen content of Pavetta assimilis plants grown in nitrogen-free sand for 98 days did not provide unequivocal proof of nitrogen fixation because control plants which are known to be unable to fix nitrogen were not included in the experiment as a check on possible contamination by combined nitrogen during the protracted experimental period. c) The low rates of acetylene reduction (5,1 and 4,4 nl g-l h-1 for nodulated leaves of Pavetta assimilis and P. zeyheri respectively) were based on a few determinations which were made when we were still relatively unfamiliar with the pitfalls of the acetylene reduction method.
Materials and Methods Acetylene reduction: Well nodulated leaves of Pavetta assimilis SOND., P. assimilis SOND. var. pubescens BREM., P. barbertonensis BREM., P. cooperi HARV. & SOND., P. eylesii S. MooRE, P. edentula SOND. ex HARV & SOND., P. gracilifolia BREM., P. lanceolata ECKL., P. revoluta HOCHST., P. schumanniana F. HOFFM., P. tristis BREM., P. zeyheri SOND., Psychotria kirkii HIERN. and P. punctata VATKE (= Psychotria bacteriophila VAL.) were used. The experiment was repeated at three different times, viz. in spring (September-October); in mid-summer (December-January) and in the autumn (April-May). Young developing leaves and mature leaves were used in separate treatments during the spring and summer trials whereas mature and senescent leaves, which were partly yellowing, were tested in the autumn. The freshly excised leaves (usually about 3 g) were lightly rolled up and inserted into a 50 ml Ultra Asept syringe. When dealing with large leaves such as those of P. edentula, the leaves were cut into strips before they were rolled up. Water was added to the syringe until it covered the leaves. Air and excess water were expelled from the syringe by compression to a volume of 20 ml - care being taken not to bruise the leaves. The needle of the syringe was pressed through a rubber serum bottle stopper which sealed a 5 I all-glass container holding a mixture of 1 volume of acetylene to 4 volumes of air at a positive pressure (initial pressure 1,5 atmospheres). The acetylene used in the gas mixture was previously scrubbed in concentrated H 2S0 4 (HARDY et aI., 1968). After the syringe was filled to its maximum extent with the gas mixture, it was withdrawn from the reservoir and compressed until it contained 30 ml of the gas mixture at atmospheric pressure. The syringe was sealed with a rubber serum bottle stopper and held in an inclined position to ensure that the leaves drained completely of water still present in the syringe. In all cases the treatments were split and two of the four replicates were held in indirect sunlight whilst the other two replicates were held in darkness in the same laboratory. Temperature was not controlled. Control treatments in which leaves were incubated as described above, but in air instead of an acetylene-air mixture were set up for all the above treatments. The syringes were charged with the acetylene-air mixture in a fume cupboard in order not to contaminate the laboratory air with acetylene. After 1, 6 and 24 hours of incubation, 1 ml of the gas in each syringe was analyzed by gas chromatography for its ethylene content. The ethylene content of 1 ml samples of the laboratory air and the acetylene-air mixture was also repeatedly determined. For the ethylene determinations a Beckman GC-5 apparatus fitted with a 3 m stainless steel column with internal diameter of 3 mm was used. The column was packed with Porapak R (Waters Assoc., Inc.). The Column temperature was 60° C and the flow rate of the
z.
Pflanzenphysiol. Ed. 73. S. 103-108. 1974.
Nitrogen Fixation of Pavetta and Psycho tria
105
helium carrier gas was 30 ml min-i. A flame ionization detector was used. The results were recorded on a 20 cm Beckman recorder which ran at a speed of 2,5 cm min-i. The 1 ml gas syringe was kept in an evacuated flask for 15 minutes between injections of the gas chromatograph. All the analyses were carried out at an attenuation of 2 X 10. The ethylene peak was recorded 2,2 minutes after injection and was well separated from the excess of acetylene which had a longer retention time. After incubation, the dry weight (0,5-1,1 g) and total nitrogen content (11-30 mg) of the leaves were determined - the latter by the micro-Kjeldahl method.
Growth studies The following species were experimented on: Pavetta assimilis, P. eylesii, P. lanceolata, P. schumanniana, Psycho tria kirkii, P. punctata and Citrus nobilus LOUR. var. deliciosa, cult. Cleopatra (Cleopatra mandarin). Seeds of the Pavetta species were collected in the field. The seed of P. kirkii were obtained from Dr. MADY DE BONT of Lovanium University, Kinshasa, ZaIre and those of P. punctata from the Fairchild Tropical Gardens, Miami, Florida, U.S.A. The citrus seed was obtained from D.F.A. von Staden, Dept. of Horticulture, University of Pretoria. The complete nutrient solution used contained 705 mg Ca(N03)2·4H20, 1020 mg KN0 3, 350 mg KH2P0 4, 490 mg MgS0 4 ·7H20, 24 mg FeCl3·6H20, 0,286 mg H 3B03, 0,181 mg MnCl 2·4H20, 0,008 mg CuS04·5H2,O, 0,022 mg ZnS04·7H20 and 0,009 mg H 2Mo0 4 per 4 litre of solution. In the nitrogen-free culture solutions the nitrates were replaced by equimolar amounts of chlorides. The pH of both culture solutions was initially adjusted to 6 by the addition of 1 N NaOH. The seeds were germinated and the seedlings grown in sand for 10 weeks in a glasshouse in which the temperature was maintained at 27° C during the day and 21 ° C at night. The sand was flushed twice weekly with the complete nutrient solution and moistened in between with tap water. Two similar groups of 12 well-nodulated seedlings each were selected for each species. The fresh weight, dry weight and total nitrogen content of the individual plants of one of each pair of groups were determined. The seedlings of the remaining groups were transferred to nitrogen-free culture solutions. In the case of some species a few additional seedlings, comparable to the ones used in the nitrogen-free culture solution, were simultaneously transferred to a complete culture solution. The plants were grown singly in 1 litre polystyrene pots which each contained 880 ml of culture solution. The seedlings were supported by cotton wool plugs in the holes of the tightly fitting lids of the pots. Distilled water was added to the pots at weekly intervals to maintain a relatively constant volume of the nutrient solution. Leaves that abscised during the experimental period were collected as far as this was possible. After the plants had grown in the culture solutions for 75 days they were processed in the same way as described above. All nitrogen analyses were done in duplicate, at least, by the micro-KJELDAHL method.
Results
Acetylene reduction: After allowance had been made for both the ethylene originally present in the incubation medium and the ethylene produced by the leaves in the abscence of acetylene, no evidence of acetylene reduction could be detected in any of the species in any of the treatments.
z.
P/lanzenphysiol. Bd. 73. S. 103-108. 1974.
106
N.
GROBBELAAR
Cleo
+
N
and E.
G. GROENEWALD
Cleo -N
Psy. bac.
+N
Psy. bac. -N
Fig. 1: Photograph of seedlings after having grown in culture solution for 70 days. From left to right: Citrus (Cleopatra mandarin) with and without combined nitrogen in the culture solution, Psychotria punctata (= P. bacteriophylla) with and without combined nitrogen in the culture solution. Table 1: Mean dry weights and nitrogen contents of comparable plants before and after growth for 75 days in a nitrogen-free culture solution. Species
Citrus Pav. assimilis Pav. eylesii Pav. lanceolata Pav. schumanniana Psy. kirkii Psy. punctata
Dry weight/pIt (mg) Initial Final 135,3 96,4 75,3 36,9 59,4 128,5 41,8
271,1 198,2 184,3 121,1 244,2 220,5 134,0
Initial 2,57 2,16 1,67 0,98 1,75 1,84 0,77
N content/pit (mg) Final Loss or gain 2,46 2,15 1,63 1,08 1,90 1,59 0,82
-0,11 -0,01 -0,04 + 0,10 + 0,15 -0,25 + 0,05
Growth studies: The plants of all species developed severe symptoms of nitrogen deficiency whilst growing in the nitrogen-free culture solution despite the presence of numerous bacteria-filled nodules in their leaves (see fig. 1). Indeed, the experiment had to be terminated after 75 days because of the approaching death of some of the plants in this treatment. As can be seen from table 1, the mean nitrogen content of the plants remained virtually constant during the experimental period. In no case did the initial and final mean nitrogen content of the plants differ significantly even at the 30 0J0 level of probability when tested statistically be means of the t-test. Despite the low level of combined nitrogen in the complete nutrient solution, the plants that grew in it had a healthy appearance and grew much more vigorously than their counterparts in the nitrogen-free culture solution (see fig. 1). Discussion and Conclusions The results of this study, on which we place more reliance than our earlier work et aI., 1971), do not support the contention that the bacteria-filled leaf nodules of Pavetta or Psycho tria species normally fix elementary nitrogen. The
(GROBBELAAR
Z. Pflanzenphysiol. Bd. 73. S. 103-108. 1974.
Nitrogen Fixation of Pavetta and Psycho tria
107
present results are consistent with several less ambitious experiments that we have carried out since our earlier report. The use of the KJELDAHL method in determining gains in nitrogen is a relatively insensitive way of detecting nitrogen fixation partly because of its inherent insensitivity and also because determinations have to be performed on different sets of plants. These defects can, however, be compensated for by permitting the plants to fix nitrogen over an extended period. Despite precautionary measures, gains in nitrogen determined in this way can still be the result of contamination rather than fixation. It is therefore essential, especially in cases where low rates of nitrogen fixation are anticipated, to check the possibility of contamination by incorporating plants in the experiment which are generally regarded as being non-fixers of nitrogen. Citrus seedlings were used for this purpose in the present study. Since the nitrogen content of the Pavetta and Psycho tria plants did not increase significantly, the results of the Citrus plants are superfluous in arriving at the conclusion that the test plants did not fix nitrogen. Although we do not know how to account for the gain in the nitrogen content of P. assimilis that we observed earlier in a similar experiment (GROBBELAAR et aI., 1971), those results, as explained above, do not provide unequivocal evidence for the ability of P. assimilis plants to fix nitrogen. For the same reasons the interpretation by VON FABER (1912, 1914) and SILVER et aI. (1963) of their results concerning the nitrogen fixing ability of nodulated Pavetta and/or Psychotria plants should provisionally be treated with scepticism. The 15N-method is probably the most sensitive and reliable available direct method of testing an organism's ability to fix nitrogen. Unfortunately it is not, as in the present case, always feasible to expose the test material to a UN-enriched atmosphere for extended periods without causing deleterious effects to the system under investigation. Very low rates of nitrogen fixation can therefore not always be measured by this method. In the absence of positive results for nitrogen fixation by a direct method, positive results by the acetylene reduction method are not a satisfactory substitute. Because acetylene is apparently universally reduced to ethylene by nitrogenase, a negative result with the acetylene-reduction method which is up to 1000 times more sensitive than the 15N-method (HARDY et aI., 1968), probably constitutes the most convincing way of discrediting the occurrence of nitrogen fixation. The only positive results that were obtained by the acetylene-reduction method with nodulated leaves of Pavetta or Psychotria species appear to be the very weakly positive ones recorded by GROBBELAAR et aI. (1971). From several subsequent assays in addition to the results of the more comprehensive experiment recorded in this paper, all of which consistently yielded negative results, we are now convinced that the earlier results should be rejected in favour of the latter. Z. P/lanzenphysiol. Bd. 73. S. 103-108. 1974.
108
N. GROBBELAAR and E. G. GROENEWALD
Acknowledgements This work was supported by a grant from the Department of Agricultural Technical Services of the Republic of South Africa.
References BECKING, J. H.: Plant and Soil, Special Vol. 361 (1971). BETTELHEIM, K. A., et al.: J. gen. Microbiol. 54, 177 (1968). BOND, G.: Nature 182, 474 (1958). BREMEKAMP, C. E. B.: J. Botany, London 71, 271 (1933). - Repert. Spec. nov. regno vegetabilis 37, 1 (1934). GROBBELAAR, N., et al.: Plant and Soil, Special Vol. 325 (1971). HARDY, A. W. F., et al.: Plant Physiol. 43, 1185 (1968). HUMM, H. J.: J. N. Y. Botan. Garden 45, 193 (1944). RAo, K. A.: Agric. J. India 18, 132 (1923). SILVER, W. S., et al.: Nature 199, 396 (1963). SILVESTER, W. B., and ASTRIDGE, S.: Plant and Soil 35, 647 (1971). VON FABER, F. C.: Jahrb. Wiss. Botan. 51, 285 (1912). - Jahrb. Wiss. Botan. 54, 243 (1914). N. GROBBELAAR and E. G. GROBBELAAR, Margaretha Mes Institute for Plant Physiology and Biochemistry, University of Pretoria, Republic of South Africa.
z. Pjlanzenphysiol. Bd. 73. S.
103-108. 1974.
Nitrogen Fixation by Nodulated Species of Pavetta and Psychotria N. GROBBELAAR and E. G. GROENEWALD With 1 figure Received January 9, 1974
Summary Leaves bearing bacteria-filled nodules from seven Pavetta and two Psychotria species were tested for nitrogenase activity by the acetylene reduction method for up to 24 hours with negative results. Four of the Pavetta species and the two Psychotria species used in the acetylene reduction tests were also grown in a nitrogen-free culture solution for 75 days. The plants developed severe symptoms of nitrogen deficiency and no increase in their nitrogen content could be detected during the experimental period.
Key words: Leaf nodules; nitrogen fixation; Pavetta; Psychotria.
Introduction
Bacterial leaf nodules are a common feature of many species of Pavetta and Psychotria (BREMEKAMP, 1933, 1934). There is considerable uncertainty about the nitrogen fixing ability of such plants. None of the reports (VON FABER, 1912, 1914; SILVER et aI., 1963; GROBBELAAR et aI., 1971) which mentions positive results for nitrogen fixation is convincing. In some cases (VON FABER, 1912, 1914; RAO, 1923; SILVER et aI., 1963; BETTELHEIM et aI., 1968) it is claimed that the endophyte was isolated and shown to be able to fix nitrogen. However, in no case was it unequivocally established that the isolate was the true endophyte of the leaf nodules. To add to the uncertainty, BOND (1958), BECKING (1971) and SILVESTER and ASTRIDGE (1971), using sensitive modern methods, were unable to demonstrate nitrogen fixation in nodulated species for which other workers reported positive results. HUMM (1944) also did not find evidence for nitrogen fixation in experiments with Psycho tria punctata. In view of the above, we have now more rigorously investigated a wider range of nodulated species for their ability to fix nitrogen. We were prompted to undertake the present study because in our initial investigation (GROBBELAAR et aI., 1971) a) The UN-method, of which we have had considerable experience, gave negative results when applied to nodulated leaves as well as whole seedlings of Pavetta assimilis ander various conditions.
z.
Pflanzenphysiol. Bd. 73. S. 103-108. 1974.
104
N. GROBBELAAR and E. G. GROENEWALD
b) The small increase (7 mg) in the nitrogen content of Pavetta assimilis plants grown in nitrogen-free sand for 98 days did not provide unequivocal proof of nitrogen fixation because control plants which are known to be unable to fix nitrogen were not included in the experiment as a check on possible contamination by combined nitrogen during the protracted experimental period. c) The low rates of acetylene reduction (5,1 and 4,4 nl g-l h-1 for nodulated leaves of Pavetta assimilis and P. zeyheri respectively) were based on a few determinations which were made when we were still relatively unfamiliar with the pitfalls of the acetylene reduction method.
Materials and Methods Acetylene reduction: Well nodulated leaves of Pavetta assimilis SOND., P. assimilis SOND. var. pubescens BREM., P. barbertonensis BREM., P. cooperi HARV. & SOND., P. eylesii S. MooRE, P. edentula SOND. ex HARV & SOND., P. gracilifolia BREM., P. lanceolata ECKL., P. revoluta HOCHST., P. schumanniana F. HOFFM., P. tristis BREM., P. zeyheri SOND., Psychotria kirkii HIERN. and P. punctata VATKE (= Psychotria bacteriophila VAL.) were used. The experiment was repeated at three different times, viz. in spring (September-October); in mid-summer (December-January) and in the autumn (April-May). Young developing leaves and mature leaves were used in separate treatments during the spring and summer trials whereas mature and senescent leaves, which were partly yellowing, were tested in the autumn. The freshly excised leaves (usually about 3 g) were lightly rolled up and inserted into a 50 ml Ultra Asept syringe. When dealing with large leaves such as those of P. edentula, the leaves were cut into strips before they were rolled up. Water was added to the syringe until it covered the leaves. Air and excess water were expelled from the syringe by compression to a volume of 20 ml - care being taken not to bruise the leaves. The needle of the syringe was pressed through a rubber serum bottle stopper which sealed a 5 I all-glass container holding a mixture of 1 volume of acetylene to 4 volumes of air at a positive pressure (initial pressure 1,5 atmospheres). The acetylene used in the gas mixture was previously scrubbed in concentrated H 2S0 4 (HARDY et aI., 1968). After the syringe was filled to its maximum extent with the gas mixture, it was withdrawn from the reservoir and compressed until it contained 30 ml of the gas mixture at atmospheric pressure. The syringe was sealed with a rubber serum bottle stopper and held in an inclined position to ensure that the leaves drained completely of water still present in the syringe. In all cases the treatments were split and two of the four replicates were held in indirect sunlight whilst the other two replicates were held in darkness in the same laboratory. Temperature was not controlled. Control treatments in which leaves were incubated as described above, but in air instead of an acetylene-air mixture were set up for all the above treatments. The syringes were charged with the acetylene-air mixture in a fume cupboard in order not to contaminate the laboratory air with acetylene. After 1, 6 and 24 hours of incubation, 1 ml of the gas in each syringe was analyzed by gas chromatography for its ethylene content. The ethylene content of 1 ml samples of the laboratory air and the acetylene-air mixture was also repeatedly determined. For the ethylene determinations a Beckman GC-5 apparatus fitted with a 3 m stainless steel column with internal diameter of 3 mm was used. The column was packed with Porapak R (Waters Assoc., Inc.). The Column temperature was 60° C and the flow rate of the
z.
Pflanzenphysiol. Ed. 73. S. 103-108. 1974.
Nitrogen Fixation of Pavetta and Psycho tria
105
helium carrier gas was 30 ml min-i. A flame ionization detector was used. The results were recorded on a 20 cm Beckman recorder which ran at a speed of 2,5 cm min-i. The 1 ml gas syringe was kept in an evacuated flask for 15 minutes between injections of the gas chromatograph. All the analyses were carried out at an attenuation of 2 X 10. The ethylene peak was recorded 2,2 minutes after injection and was well separated from the excess of acetylene which had a longer retention time. After incubation, the dry weight (0,5-1,1 g) and total nitrogen content (11-30 mg) of the leaves were determined - the latter by the micro-Kjeldahl method.
Growth studies The following species were experimented on: Pavetta assimilis, P. eylesii, P. lanceolata, P. schumanniana, Psycho tria kirkii, P. punctata and Citrus nobilus LOUR. var. deliciosa, cult. Cleopatra (Cleopatra mandarin). Seeds of the Pavetta species were collected in the field. The seed of P. kirkii were obtained from Dr. MADY DE BONT of Lovanium University, Kinshasa, ZaIre and those of P. punctata from the Fairchild Tropical Gardens, Miami, Florida, U.S.A. The citrus seed was obtained from D.F.A. von Staden, Dept. of Horticulture, University of Pretoria. The complete nutrient solution used contained 705 mg Ca(N03)2·4H20, 1020 mg KN0 3, 350 mg KH2P0 4, 490 mg MgS0 4 ·7H20, 24 mg FeCl3·6H20, 0,286 mg H 3B03, 0,181 mg MnCl 2·4H20, 0,008 mg CuS04·5H2,O, 0,022 mg ZnS04·7H20 and 0,009 mg H 2Mo0 4 per 4 litre of solution. In the nitrogen-free culture solutions the nitrates were replaced by equimolar amounts of chlorides. The pH of both culture solutions was initially adjusted to 6 by the addition of 1 N NaOH. The seeds were germinated and the seedlings grown in sand for 10 weeks in a glasshouse in which the temperature was maintained at 27° C during the day and 21 ° C at night. The sand was flushed twice weekly with the complete nutrient solution and moistened in between with tap water. Two similar groups of 12 well-nodulated seedlings each were selected for each species. The fresh weight, dry weight and total nitrogen content of the individual plants of one of each pair of groups were determined. The seedlings of the remaining groups were transferred to nitrogen-free culture solutions. In the case of some species a few additional seedlings, comparable to the ones used in the nitrogen-free culture solution, were simultaneously transferred to a complete culture solution. The plants were grown singly in 1 litre polystyrene pots which each contained 880 ml of culture solution. The seedlings were supported by cotton wool plugs in the holes of the tightly fitting lids of the pots. Distilled water was added to the pots at weekly intervals to maintain a relatively constant volume of the nutrient solution. Leaves that abscised during the experimental period were collected as far as this was possible. After the plants had grown in the culture solutions for 75 days they were processed in the same way as described above. All nitrogen analyses were done in duplicate, at least, by the micro-KJELDAHL method.
Results
Acetylene reduction: After allowance had been made for both the ethylene originally present in the incubation medium and the ethylene produced by the leaves in the abscence of acetylene, no evidence of acetylene reduction could be detected in any of the species in any of the treatments.
z.
P/lanzenphysiol. Bd. 73. S. 103-108. 1974.
106
N.
GROBBELAAR
Cleo
+
N
and E.
G. GROENEWALD
Cleo -N
Psy. bac.
+N
Psy. bac. -N
Fig. 1: Photograph of seedlings after having grown in culture solution for 70 days. From left to right: Citrus (Cleopatra mandarin) with and without combined nitrogen in the culture solution, Psychotria punctata (= P. bacteriophylla) with and without combined nitrogen in the culture solution. Table 1: Mean dry weights and nitrogen contents of comparable plants before and after growth for 75 days in a nitrogen-free culture solution. Species
Citrus Pav. assimilis Pav. eylesii Pav. lanceolata Pav. schumanniana Psy. kirkii Psy. punctata
Dry weight/pIt (mg) Initial Final 135,3 96,4 75,3 36,9 59,4 128,5 41,8
271,1 198,2 184,3 121,1 244,2 220,5 134,0
Initial 2,57 2,16 1,67 0,98 1,75 1,84 0,77
N content/pit (mg) Final Loss or gain 2,46 2,15 1,63 1,08 1,90 1,59 0,82
-0,11 -0,01 -0,04 + 0,10 + 0,15 -0,25 + 0,05
Growth studies: The plants of all species developed severe symptoms of nitrogen deficiency whilst growing in the nitrogen-free culture solution despite the presence of numerous bacteria-filled nodules in their leaves (see fig. 1). Indeed, the experiment had to be terminated after 75 days because of the approaching death of some of the plants in this treatment. As can be seen from table 1, the mean nitrogen content of the plants remained virtually constant during the experimental period. In no case did the initial and final mean nitrogen content of the plants differ significantly even at the 30 0J0 level of probability when tested statistically be means of the t-test. Despite the low level of combined nitrogen in the complete nutrient solution, the plants that grew in it had a healthy appearance and grew much more vigorously than their counterparts in the nitrogen-free culture solution (see fig. 1). Discussion and Conclusions The results of this study, on which we place more reliance than our earlier work et aI., 1971), do not support the contention that the bacteria-filled leaf nodules of Pavetta or Psycho tria species normally fix elementary nitrogen. The
(GROBBELAAR
Z. Pflanzenphysiol. Bd. 73. S. 103-108. 1974.
Nitrogen Fixation of Pavetta and Psycho tria
107
present results are consistent with several less ambitious experiments that we have carried out since our earlier report. The use of the KJELDAHL method in determining gains in nitrogen is a relatively insensitive way of detecting nitrogen fixation partly because of its inherent insensitivity and also because determinations have to be performed on different sets of plants. These defects can, however, be compensated for by permitting the plants to fix nitrogen over an extended period. Despite precautionary measures, gains in nitrogen determined in this way can still be the result of contamination rather than fixation. It is therefore essential, especially in cases where low rates of nitrogen fixation are anticipated, to check the possibility of contamination by incorporating plants in the experiment which are generally regarded as being non-fixers of nitrogen. Citrus seedlings were used for this purpose in the present study. Since the nitrogen content of the Pavetta and Psycho tria plants did not increase significantly, the results of the Citrus plants are superfluous in arriving at the conclusion that the test plants did not fix nitrogen. Although we do not know how to account for the gain in the nitrogen content of P. assimilis that we observed earlier in a similar experiment (GROBBELAAR et aI., 1971), those results, as explained above, do not provide unequivocal evidence for the ability of P. assimilis plants to fix nitrogen. For the same reasons the interpretation by VON FABER (1912, 1914) and SILVER et aI. (1963) of their results concerning the nitrogen fixing ability of nodulated Pavetta and/or Psychotria plants should provisionally be treated with scepticism. The 15N-method is probably the most sensitive and reliable available direct method of testing an organism's ability to fix nitrogen. Unfortunately it is not, as in the present case, always feasible to expose the test material to a UN-enriched atmosphere for extended periods without causing deleterious effects to the system under investigation. Very low rates of nitrogen fixation can therefore not always be measured by this method. In the absence of positive results for nitrogen fixation by a direct method, positive results by the acetylene reduction method are not a satisfactory substitute. Because acetylene is apparently universally reduced to ethylene by nitrogenase, a negative result with the acetylene-reduction method which is up to 1000 times more sensitive than the 15N-method (HARDY et aI., 1968), probably constitutes the most convincing way of discrediting the occurrence of nitrogen fixation. The only positive results that were obtained by the acetylene-reduction method with nodulated leaves of Pavetta or Psychotria species appear to be the very weakly positive ones recorded by GROBBELAAR et aI. (1971). From several subsequent assays in addition to the results of the more comprehensive experiment recorded in this paper, all of which consistently yielded negative results, we are now convinced that the earlier results should be rejected in favour of the latter. Z. P/lanzenphysiol. Bd. 73. S. 103-108. 1974.
108
N. GROBBELAAR and E. G. GROENEWALD
Acknowledgements This work was supported by a grant from the Department of Agricultural Technical Services of the Republic of South Africa.
References BECKING, J. H.: Plant and Soil, Special Vol. 361 (1971). BETTELHEIM, K. A., et al.: J. gen. Microbiol. 54, 177 (1968). BOND, G.: Nature 182, 474 (1958). BREMEKAMP, C. E. B.: J. Botany, London 71, 271 (1933). - Repert. Spec. nov. regno vegetabilis 37, 1 (1934). GROBBELAAR, N., et al.: Plant and Soil, Special Vol. 325 (1971). HARDY, A. W. F., et al.: Plant Physiol. 43, 1185 (1968). HUMM, H. J.: J. N. Y. Botan. Garden 45, 193 (1944). RAo, K. A.: Agric. J. India 18, 132 (1923). SILVER, W. S., et al.: Nature 199, 396 (1963). SILVESTER, W. B., and ASTRIDGE, S.: Plant and Soil 35, 647 (1971). VON FABER, F. C.: Jahrb. Wiss. Botan. 51, 285 (1912). - Jahrb. Wiss. Botan. 54, 243 (1914). N. GROBBELAAR and E. G. GROBBELAAR, Margaretha Mes Institute for Plant Physiology and Biochemistry, University of Pretoria, Republic of South Africa.
z. Pjlanzenphysiol. Bd. 73. S.
103-108. 1974.