- Email: [email protected]
Preparation of submicrogram nitrogen samples for isotope analysis by the GS1 emission spectrometer
International J¢mrnul c~f Applied Radialion and I.~ropes Voi. 32. pp. 215 to 217, 1981 Printed in Great Britain. All rights reserved
0020-?08X/81/040215-03502.00/0 Copyright ~ 1981 Pergamon Press Lid
Preparation of Submicrogram Nitrogen Samples for Isotope Analysis by the GS1 Emission Spectrometer FRANCIS
MARTIN, BERNARD
MAUDINAS,
MICHEL
CHEMARDIN
and PIERRE GADAL Laboratoire de Biologie V6g~tale, E.R.A. No. 799 C.N.R.S., Universit6 de Nancy I C.O. 140 54037 Nancy-cedex, France
(Received 17 September 1980) A convenient method is described for the preparation of submicrogram nitrogen electrodeless discharge tubes without the addition of noble gas. The stable nitrogen isotope analysis was carried out by using the GS 1 emission spectrometer whose possibilities have been tested. The preparative method described is particularly fitted for the analysis of biological samples having a low t sN enrichment.
Introduction MANY spectrometric methods have been reported for measuring nitrogen-15 enrichment. Routinely, the size of nitrogen samples analysed in electrodeless discharge tubes has scarcely been lower than 1 to 5/zg. Therefore, experimental devices are needed for the study of nitrogen uptake and assimilation by plants in order to determine the nolsN excess in samples containing submicrogram quantities of nitrogen. Many authors "'2) showed that noble gases sustained a discharge of submicrogram quantities of nitrogen gas in discharge tubes. In this work is developed a preparation method for the determination of submicrogram N quantities in discharge tubes without the sustaining of noble gases. The nitrogen isotopes analysis were carried out by using a new commercially available emission spectrometer GS 1 whose properties have been tested here.
go back. This procedure gives an accurate estimation of the base line in a unique scan. The region of the discharge tubes excited by a high frequency source was located between two electrodes separated by a 20 mm distance. These conditions allowed the use of short discharge tubes.
Discharge tube preparation
The discharge tubes were prepared according to Fiedler and Proksch. (s) After determination of total nitrogen by nesslerization, an aliquot of (NH,),SO4 solution was taken in a capillary tube (I.D. = 1 ram, O.D. = 2 ram, length = 10 ram), and dried under an infrared lamp. The (NH4hSO4 concentration was chosen to give a final nitrogen content of 0.4 to 0.5 pg in the capillary tube. In order to convert the ammonium and to trap the contaminant gases, C a n pieces previously heated overnight in a furnace at 1000°C and CuO wires were used. The C a n pieces were first introduced at the bottom of a Pyrex glass tube (I.D. 2.6 ram, O.D. 4.0 turn, length 100 ram), then the CuO wires in the M a t e r i a l s and M e t h o d s middle and finally the small capillary tube containing Apparatus the sample was added near the open extremity. This When nitrogen is excited in an electrodeless dis- tube was further connected to a 40ram Ultravidecharge tube by a high frequency generator, the helium tubing (Verneret) branched horizontally to a emitted light spectra differ for the three isotopic mol- high vacuum line. The system was then evacuated ecules I*N~, l*N1SN, ISN~ and the bands heads are with a handtorch plus Edwards Tesla coil around 10-~torr and the tube vertically positioned was at 297,68. 298,29 and 298.86nm respectively. The sealed off at a length of approximately 30 mm from spectrometer used in this study is the G S I manufactured by Sopra {France). In this apparatus, a fixed the bottom. The tubes must be gently sealed off to prevent overheating of the nitrogen sample. Furtherconcave grating (2400 lines ram-l blazed for 300 nm) is used as the light scattering system and the above more this sealed discharge tube was soldered to an wavelengths are scanned by the rotation of a parallel additional Pyrex tube of the same diameter to permit glass slide.Due to an original convenient device, part an easier positioning in the electrodes compartment of the emission spectrum of nitrogen isotopes in the of the emission spectrometer. Finally, the discharge 3 0 0 n m region is scanned first in I0 s with an un- tubes were heated overnight at 570~C. Consequently, amplified go straight, and then, with a 20 s amplified the discharge tubes had a 0.3 ml volume and the 215
F. Martin et al.
216
nitrogen content was sufficient to sustain a pressure of 2 mm Hg without any noble gas addition. The lSN content of the standard discharge tubes used in this study to test the properties of the GS 1 emission spectrometer was determined by mass spectrometry. During all operations described above, great care has to be taken to keep the system free from fine dust and other impurities. Consequently the following precautions were taken:
Txa~ 1. Determination of the atom %LSN values by using the regression equation y = 0,965 x - 0.098
(1) prior to use. Pyrex tubes were washed with a sulfochromic mixture, thoroughly rinsed with bidistilled water, then heated overnight at 600°C; {2) handled either with gloves or forceps, sterile capillary tubes were used; (3) during the drying and discharge tubes preparation, the sample capillary tubes were kept in a Petri dish with concentrated sulfuric acid to trap water and possible ammonia traces. (4) finally, chemical reagents, capillaries and clean Pyrex tubes were stored under vacuum in a dessicator to prevent carbon dioxide or water absorption.
TABLE 2. "Short term" variations of standard ~SN discharge tubes with the GS I nitrogen 15 analyser. Values are in atom °g~SN
The degree of contamination which can arise has been investigated by using a (~SNHahSO, standard solution previously determined by mass spectrometry, as illustrated above :--atom °/~SN" o . mass spectrometry: 32.23. Dumas combustion and emission spectrometry: 32.72 (The values are the average of four discharge tubes). A high labeling solution was used in order to detect even traces of taN contamination in the sample, which clearly shown that no contamination had occurred during the preparation steps.
Mass spectrometry values
Emission spectrometry values Expzrimental
Corrected
0.366 0.435 0.670 1.857
0.482 0.547 0.813 2.013
0.367 0.429 0.687 1.845
Measured atom QgtSN Standards, atom °,glSN 0.368
0.442
0.670
11.033
Values 0.364 0.376 0.376 0.380 0.378 0.425 0.441 0.441 0.434 0.438 0.675 0.664 0.657 0.677 0.653 I1.103 11.250 11.115 11.169 11.224
Mean
SD°/o*
0.375
0.50
0.436
1.37
0.665
1.43
11.172
0.52
* SD°o = standard deviation. Results and Discussion A particular effort has been made concerning the methodology to provide maximal sensitivity and accuracy of nitrogen analysis in the range from natural abundance to a 10°:~atom tSN as these values encompass most of the physiological t SN studies.
Nitrogen-I: analyser In the initial stages of investigation a lot of various spectral lSN analysers, constructed by workers using devices of their own have been used. At present several types of apparatus designed excinsively for t SN spectroscopy analysis are available on the market. Among the representative instruments are the spectrometer made by Jasco (Japan) and the Statron models made by Isocomerz Co. (East Germany). Good performances were available with the Jasco N-15 emission spectrometeP a~ but some modifications are necessary on the Statron analyser.~s~ Recently, a new emission spectrometer GS 1 {Sopra, France) has been commercialized: the first part of this paper is to test its possibilities.
Standardization Above 2~0 atom ~SN a perfect concordance exists between the data given by mass spectrometry and emission spectrometry. However in the 0.366-2.0% atom ~SN range the standard curve was not linear. Nevertheless, by using a linear regression equation of the form y -- mx + b, the determination of the actual ~SN values is accessible. Thus, the equation y= 0.965 x -0.098 gives standard values within __ 2.5°:~ in all cases (Table I).
Repeatability and accuracy The results of isotopic enrichment measurements in four standard discharge tubes are shown in Table 2. Five successive scans showed that the GS 1 spectrometer gave good accuracy values for the standards even for very low isotopic excess (less than 0.10o). However, the standard deviations given in Table 2 are related to the ~short term" precision since the results were obtained successively in the course of about 30 rain. The "long term" variations due to environmental change conditions, electronic instabilities etc. were investigated for the GS I by measuring samples
217
Nitrogen analysis by the GS I emission spectrometer
TABLE 3. "Long-term" variations of ~SN standard discharge tubes with the GS 1 nitrogen-15 anaiyser. Values are in atom ~tSN Hour
Measured atom ?.otSN
0 2 4 6 Mean SD "o
0.362 0.356 0.376 0.366 0.365 2.0
Day
0.431 0.423 0.441 0.436 0.433 1.5
0.633 0.639 0.621 0.627 0.630 1.1
1.961 2.061 2.053 1.997 2.018 2.0
Measured atom !..,isN Values
10.846 10.788 11.170 10.663 10.867 1.72
Mean SD ~
0.462 0.456 0.477 0.458 0.463 1.77
0.622 0.622 0.635 0.627 0.626 0.85
3.469 3.464 3.478 -3.470 0.167
11.013 10.983 10.718 10.772 10.871 1.18
Measured atom 0otSN
0 2 3 4 5 Mean SD°o
0.351 0.369 0.353 0.362 0.366 0.360 2.0
0.433 0.431 0.438 0.448 0.421 0.434 2.0
0.630 0.636 0.625 0.627 0.640 0.632 0.9
1.973 1.965 2.024 2.050 1.990 2.000 1.6
11.200 10.666 10.810 11.024 11.164 10.972 1.9
of different isotopic abundance over a day course and during a week period (Table 3). The results showed that the "long term" and the "short term" variations were of the same order. Both vary between 1.5 and 2.3?/0 for ~SN excess lower than 1% and between 0.6 and 2.5?/0 up to 10% excess, This accuracy is similar to the one obtained by authors using other commercial apparatus/"'6"~) In addition, our results confirm previous data published recently on the GS 1 prototype: s) Discharge tubes properties
The prepared discharge tubes, containing about 0.5/~g N in 0.3 ml volume emitted, when excited, a bright nitrogen discharge that lasted at least 15 rain. The lifetime of nitrogen emission was long enough to obtain accurate results in nitrogen- 15 analysis (Table 4) without being forced to use the tedious and expensive noble gas supplementation. However, possible nitrogen contamination could occur at any point in the analytical procedure, and therefore great care in manipulation is essential (see Materials and Methods). Biological samples analysis
A convenient system to study the nitrogen metabolism in corn seedlings has been developed by using mature sections and root tips (5 mm length). (e) In this study, 20root sections were incubated for 3 h in 5 m M KlSNOs (Atom 9~,otSN). Despite the very low amount of nitrogen present, we have been able, according to the method described above, to follow the incorporation of l SN in the fraction soluble in C H 3 O H - C H C I 3 - H 2 0 (12:5:3, v/v). Each sample has
A.R,n. 32 4
T~t.E 4. Repeatability of measurements of "short" discharge tubes (corrected values in atom ~tSN)
e
been duplicated and the light emission was bright and stable over a long period of time. The atom To i sN were 2.499 and 3.762 in mature sections and tips respectively. The accuracy (SDTo 1.5) and the repeatability ( + 0.36%) are indeed satisfactory. In agreement with previous data concerning the determination of enzymatic activities°°) the NO~ incorporation in corn root tips is much faster than in mature sections. In conclusion, the preparative method described for biological samples having a low l SN enriduncnt often a good tool by the use of GS 1 spectrometer for the study of nitrogen metabolism in plants. Acknowledgements--The authors thank Dr G. Guiraud for providing the standard samples and Mr J. I- Stehl~ for expert assistance. F.M. thanks the British Council for financial ,support during his slay in the Biochemistry Department of Rothamsted Experiments] Station. He is particularly indebted to Dr B. Miflin for his welcome and to Dr J. Hill for his helpful edvices during his training in the emission spectrometry techniques. This investigation was partly supported by a grant from CNRS (ATP No. 4113, RCP No. 389) and DGRST (AC No. 7870445).
References 1. MUHAtIMADS. and KUMAZ^W^K. Z. Pflanzenernaehr. Badenkd. 8, 529-536 (1976). 2. GOL~ J. A, and MIDDEI.BOE V. Anal. Chim, Acta 43, 229-234 (1968~ 3. FW.DLeRR. and P a ~ G. Anal. Chint Acta 71, 1-61
(1975). 4. Guw,Aun G. and BUSCAaLETL. A. Int. J. Appl. Radial. Isot. 26, 187-193 (1975). 5. LLOYD-JoNi~C. P~ HUDD G. A. and HILL-COTTINGHAM D. G. Analyst 99, 580-587. (1974). 6. K~NEY D. R. and TED,,CO M. J. Anal. Chim. Acta 65, 19-34 (1973). 7. FIEDLEIt R. and PItOrsCH G. Plant and Soil 36, 371-378. (1972). 8. GUIRAUDG. and FARDI~U J. C. Analusis, 8, 148-152
(1980). 9. OAKS A, STULEN I. and BOSEL 1. Can. J. Bot. 57, 1824-1829 (1979). 10. OArS A., STULD~1., JONESK., WtNSPEAa M. J., M ~ A S. ~L ] J I L L I. L. Planta 148, 477--484 (1980).
0020-?08X/81/040215-03502.00/0 Copyright ~ 1981 Pergamon Press Lid
Preparation of Submicrogram Nitrogen Samples for Isotope Analysis by the GS1 Emission Spectrometer FRANCIS
MARTIN, BERNARD
MAUDINAS,
MICHEL
CHEMARDIN
and PIERRE GADAL Laboratoire de Biologie V6g~tale, E.R.A. No. 799 C.N.R.S., Universit6 de Nancy I C.O. 140 54037 Nancy-cedex, France
(Received 17 September 1980) A convenient method is described for the preparation of submicrogram nitrogen electrodeless discharge tubes without the addition of noble gas. The stable nitrogen isotope analysis was carried out by using the GS 1 emission spectrometer whose possibilities have been tested. The preparative method described is particularly fitted for the analysis of biological samples having a low t sN enrichment.
Introduction MANY spectrometric methods have been reported for measuring nitrogen-15 enrichment. Routinely, the size of nitrogen samples analysed in electrodeless discharge tubes has scarcely been lower than 1 to 5/zg. Therefore, experimental devices are needed for the study of nitrogen uptake and assimilation by plants in order to determine the nolsN excess in samples containing submicrogram quantities of nitrogen. Many authors "'2) showed that noble gases sustained a discharge of submicrogram quantities of nitrogen gas in discharge tubes. In this work is developed a preparation method for the determination of submicrogram N quantities in discharge tubes without the sustaining of noble gases. The nitrogen isotopes analysis were carried out by using a new commercially available emission spectrometer GS 1 whose properties have been tested here.
go back. This procedure gives an accurate estimation of the base line in a unique scan. The region of the discharge tubes excited by a high frequency source was located between two electrodes separated by a 20 mm distance. These conditions allowed the use of short discharge tubes.
Discharge tube preparation
The discharge tubes were prepared according to Fiedler and Proksch. (s) After determination of total nitrogen by nesslerization, an aliquot of (NH,),SO4 solution was taken in a capillary tube (I.D. = 1 ram, O.D. = 2 ram, length = 10 ram), and dried under an infrared lamp. The (NH4hSO4 concentration was chosen to give a final nitrogen content of 0.4 to 0.5 pg in the capillary tube. In order to convert the ammonium and to trap the contaminant gases, C a n pieces previously heated overnight in a furnace at 1000°C and CuO wires were used. The C a n pieces were first introduced at the bottom of a Pyrex glass tube (I.D. 2.6 ram, O.D. 4.0 turn, length 100 ram), then the CuO wires in the M a t e r i a l s and M e t h o d s middle and finally the small capillary tube containing Apparatus the sample was added near the open extremity. This When nitrogen is excited in an electrodeless dis- tube was further connected to a 40ram Ultravidecharge tube by a high frequency generator, the helium tubing (Verneret) branched horizontally to a emitted light spectra differ for the three isotopic mol- high vacuum line. The system was then evacuated ecules I*N~, l*N1SN, ISN~ and the bands heads are with a handtorch plus Edwards Tesla coil around 10-~torr and the tube vertically positioned was at 297,68. 298,29 and 298.86nm respectively. The sealed off at a length of approximately 30 mm from spectrometer used in this study is the G S I manufactured by Sopra {France). In this apparatus, a fixed the bottom. The tubes must be gently sealed off to prevent overheating of the nitrogen sample. Furtherconcave grating (2400 lines ram-l blazed for 300 nm) is used as the light scattering system and the above more this sealed discharge tube was soldered to an wavelengths are scanned by the rotation of a parallel additional Pyrex tube of the same diameter to permit glass slide.Due to an original convenient device, part an easier positioning in the electrodes compartment of the emission spectrum of nitrogen isotopes in the of the emission spectrometer. Finally, the discharge 3 0 0 n m region is scanned first in I0 s with an un- tubes were heated overnight at 570~C. Consequently, amplified go straight, and then, with a 20 s amplified the discharge tubes had a 0.3 ml volume and the 215
F. Martin et al.
216
nitrogen content was sufficient to sustain a pressure of 2 mm Hg without any noble gas addition. The lSN content of the standard discharge tubes used in this study to test the properties of the GS 1 emission spectrometer was determined by mass spectrometry. During all operations described above, great care has to be taken to keep the system free from fine dust and other impurities. Consequently the following precautions were taken:
Txa~ 1. Determination of the atom %LSN values by using the regression equation y = 0,965 x - 0.098
(1) prior to use. Pyrex tubes were washed with a sulfochromic mixture, thoroughly rinsed with bidistilled water, then heated overnight at 600°C; {2) handled either with gloves or forceps, sterile capillary tubes were used; (3) during the drying and discharge tubes preparation, the sample capillary tubes were kept in a Petri dish with concentrated sulfuric acid to trap water and possible ammonia traces. (4) finally, chemical reagents, capillaries and clean Pyrex tubes were stored under vacuum in a dessicator to prevent carbon dioxide or water absorption.
TABLE 2. "Short term" variations of standard ~SN discharge tubes with the GS I nitrogen 15 analyser. Values are in atom °g~SN
The degree of contamination which can arise has been investigated by using a (~SNHahSO, standard solution previously determined by mass spectrometry, as illustrated above :--atom °/~SN" o . mass spectrometry: 32.23. Dumas combustion and emission spectrometry: 32.72 (The values are the average of four discharge tubes). A high labeling solution was used in order to detect even traces of taN contamination in the sample, which clearly shown that no contamination had occurred during the preparation steps.
Mass spectrometry values
Emission spectrometry values Expzrimental
Corrected
0.366 0.435 0.670 1.857
0.482 0.547 0.813 2.013
0.367 0.429 0.687 1.845
Measured atom QgtSN Standards, atom °,glSN 0.368
0.442
0.670
11.033
Values 0.364 0.376 0.376 0.380 0.378 0.425 0.441 0.441 0.434 0.438 0.675 0.664 0.657 0.677 0.653 I1.103 11.250 11.115 11.169 11.224
Mean
SD°/o*
0.375
0.50
0.436
1.37
0.665
1.43
11.172
0.52
* SD°o = standard deviation. Results and Discussion A particular effort has been made concerning the methodology to provide maximal sensitivity and accuracy of nitrogen analysis in the range from natural abundance to a 10°:~atom tSN as these values encompass most of the physiological t SN studies.
Nitrogen-I: analyser In the initial stages of investigation a lot of various spectral lSN analysers, constructed by workers using devices of their own have been used. At present several types of apparatus designed excinsively for t SN spectroscopy analysis are available on the market. Among the representative instruments are the spectrometer made by Jasco (Japan) and the Statron models made by Isocomerz Co. (East Germany). Good performances were available with the Jasco N-15 emission spectrometeP a~ but some modifications are necessary on the Statron analyser.~s~ Recently, a new emission spectrometer GS 1 {Sopra, France) has been commercialized: the first part of this paper is to test its possibilities.
Standardization Above 2~0 atom ~SN a perfect concordance exists between the data given by mass spectrometry and emission spectrometry. However in the 0.366-2.0% atom ~SN range the standard curve was not linear. Nevertheless, by using a linear regression equation of the form y -- mx + b, the determination of the actual ~SN values is accessible. Thus, the equation y= 0.965 x -0.098 gives standard values within __ 2.5°:~ in all cases (Table I).
Repeatability and accuracy The results of isotopic enrichment measurements in four standard discharge tubes are shown in Table 2. Five successive scans showed that the GS 1 spectrometer gave good accuracy values for the standards even for very low isotopic excess (less than 0.10o). However, the standard deviations given in Table 2 are related to the ~short term" precision since the results were obtained successively in the course of about 30 rain. The "long term" variations due to environmental change conditions, electronic instabilities etc. were investigated for the GS I by measuring samples
217
Nitrogen analysis by the GS I emission spectrometer
TABLE 3. "Long-term" variations of ~SN standard discharge tubes with the GS 1 nitrogen-15 anaiyser. Values are in atom ~tSN Hour
Measured atom ?.otSN
0 2 4 6 Mean SD "o
0.362 0.356 0.376 0.366 0.365 2.0
Day
0.431 0.423 0.441 0.436 0.433 1.5
0.633 0.639 0.621 0.627 0.630 1.1
1.961 2.061 2.053 1.997 2.018 2.0
Measured atom !..,isN Values
10.846 10.788 11.170 10.663 10.867 1.72
Mean SD ~
0.462 0.456 0.477 0.458 0.463 1.77
0.622 0.622 0.635 0.627 0.626 0.85
3.469 3.464 3.478 -3.470 0.167
11.013 10.983 10.718 10.772 10.871 1.18
Measured atom 0otSN
0 2 3 4 5 Mean SD°o
0.351 0.369 0.353 0.362 0.366 0.360 2.0
0.433 0.431 0.438 0.448 0.421 0.434 2.0
0.630 0.636 0.625 0.627 0.640 0.632 0.9
1.973 1.965 2.024 2.050 1.990 2.000 1.6
11.200 10.666 10.810 11.024 11.164 10.972 1.9
of different isotopic abundance over a day course and during a week period (Table 3). The results showed that the "long term" and the "short term" variations were of the same order. Both vary between 1.5 and 2.3?/0 for ~SN excess lower than 1% and between 0.6 and 2.5?/0 up to 10% excess, This accuracy is similar to the one obtained by authors using other commercial apparatus/"'6"~) In addition, our results confirm previous data published recently on the GS 1 prototype: s) Discharge tubes properties
The prepared discharge tubes, containing about 0.5/~g N in 0.3 ml volume emitted, when excited, a bright nitrogen discharge that lasted at least 15 rain. The lifetime of nitrogen emission was long enough to obtain accurate results in nitrogen- 15 analysis (Table 4) without being forced to use the tedious and expensive noble gas supplementation. However, possible nitrogen contamination could occur at any point in the analytical procedure, and therefore great care in manipulation is essential (see Materials and Methods). Biological samples analysis
A convenient system to study the nitrogen metabolism in corn seedlings has been developed by using mature sections and root tips (5 mm length). (e) In this study, 20root sections were incubated for 3 h in 5 m M KlSNOs (Atom 9~,otSN). Despite the very low amount of nitrogen present, we have been able, according to the method described above, to follow the incorporation of l SN in the fraction soluble in C H 3 O H - C H C I 3 - H 2 0 (12:5:3, v/v). Each sample has
A.R,n. 32 4
T~t.E 4. Repeatability of measurements of "short" discharge tubes (corrected values in atom ~tSN)
e
been duplicated and the light emission was bright and stable over a long period of time. The atom To i sN were 2.499 and 3.762 in mature sections and tips respectively. The accuracy (SDTo 1.5) and the repeatability ( + 0.36%) are indeed satisfactory. In agreement with previous data concerning the determination of enzymatic activities°°) the NO~ incorporation in corn root tips is much faster than in mature sections. In conclusion, the preparative method described for biological samples having a low l SN enriduncnt often a good tool by the use of GS 1 spectrometer for the study of nitrogen metabolism in plants. Acknowledgements--The authors thank Dr G. Guiraud for providing the standard samples and Mr J. I- Stehl~ for expert assistance. F.M. thanks the British Council for financial ,support during his slay in the Biochemistry Department of Rothamsted Experiments] Station. He is particularly indebted to Dr B. Miflin for his welcome and to Dr J. Hill for his helpful edvices during his training in the emission spectrometry techniques. This investigation was partly supported by a grant from CNRS (ATP No. 4113, RCP No. 389) and DGRST (AC No. 7870445).
References 1. MUHAtIMADS. and KUMAZ^W^K. Z. Pflanzenernaehr. Badenkd. 8, 529-536 (1976). 2. GOL~ J. A, and MIDDEI.BOE V. Anal. Chim, Acta 43, 229-234 (1968~ 3. FW.DLeRR. and P a ~ G. Anal. Chint Acta 71, 1-61
(1975). 4. Guw,Aun G. and BUSCAaLETL. A. Int. J. Appl. Radial. Isot. 26, 187-193 (1975). 5. LLOYD-JoNi~C. P~ HUDD G. A. and HILL-COTTINGHAM D. G. Analyst 99, 580-587. (1974). 6. K~NEY D. R. and TED,,CO M. J. Anal. Chim. Acta 65, 19-34 (1973). 7. FIEDLEIt R. and PItOrsCH G. Plant and Soil 36, 371-378. (1972). 8. GUIRAUDG. and FARDI~U J. C. Analusis, 8, 148-152
(1980). 9. OAKS A, STULEN I. and BOSEL 1. Can. J. Bot. 57, 1824-1829 (1979). 10. OArS A., STULD~1., JONESK., WtNSPEAa M. J., M ~ A S. ~L ] J I L L I. L. Planta 148, 477--484 (1980).