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Nitrogenase activity in the rhizosphere of Panicum virgatum
Sod B~ol. hciwn.
Vol. 7, pp 179 to 180 Pergamon Press 1975 Prmted m Great Brnam.
SHORT COMMUNICATION Nitrogenase activity in the rhizosphere of Panicum uivptum J. TJEPIXMA Department
of Biochemistry.
University
Nitrogenase activity has been previously reported in the rhizosphere of Pmicm ~nazimmz, a tropical grass (Dcbereiner er al., 1972; Dommergues et al.. 1973). I here report nitrogenase activity in a temperate species of this genus, Pmicur~l uirgatum.
Soil cores (15 cm x 5.5 cm) were taken and placed in 13 x 29 cm Saran bags of 25 pm wall thickness. Saran was used because of its very low permeability to gases (Rogers et (II., 1956). The bags were closed by twisting the ends, folding them over, and wrapping with a rubber band. Nitrogenase activity was measured by the C,H, reduction method. Gases were added to and removed from the bags by puncturing them at a point reinforced by adhesive plastic tape. The puncture was sealed by additional tape. The assays were made in air with CZH, (generated from CaC:) added to approximately 0.1 atm. Gas samples were taken at intervals with 2.5 ml disposable syringes and were stored until analyzed some hours later. The soil cores were replaced in the ground during the incubation. The gas volume in the bags was determined by injecting a known volume of CZH, or CH, into the bags at the end of the experiment. The soil cores were taken from a stand of P. cirgururc on the Curtis Prairie, a reestablished prairie at the University of Wisconsin Arboretum. Measurements of C,H, were taken at 30 and 90 min after CZHz addition, and the difference in concentration was used in calculating the rate of
Table
I. CZH, reduction
by components
Soil core
nmol C?H,; core h- ’
A B c A B C D E F D E F G H I
2.1 5.6 2.8 2.5 18.5 5.1 7.5 6.0 4.2 6.9 7.9 6.9 37 75 50
Roots and rhizomes 0.05 atm 0, Roots and rhizomes 0.20 atm O2 Soil 0.05 atm Oz Soil 020 atm 0: Intact cores Air
of soil cores Fresh wt. per core (8) 21.3 18.4 IO.7 21.3 18.4 IO.7 468 508 525 468 50x 525 613 697 665
of Wisconsin,
Madison.
WI 53706, U.S.A
CZH2 reduction. The rates were linear with time, at lcast up to 3 h after CzHz addition. the longest time measured. Yellowing of the leaves due to autumn senescence occurred during the course of the measurements. The rate of CLHz reduction about 3. 5 and 6 days before senescence ranged between IO and 16 nmol CLHZ;core h- ‘. while about 5 days after senescence the activity ranged between 28 and 32 nmol C2Hz;lcorc h- ‘. Controls ( -CIHL) showed no measurable evolution of CzH4. The rates of CzHz reduction observed arc quite small when extrapolated on a seasonal and area basis. The rates observed before senescence correspond to about I.3 kg N; ha 120 days-‘. But it is quite possible that the rates are higher earlier in the growing season. The experiment of Table I was done to dctermme whether the observed CLHz reduction was associated with the roots of P. rirgutum. The intact cores were incubated in air as above, but the other measurements were done in 123 ml bottles in mixtures of Ar and 0:. with 0.1 atm C,H,. Washed roots and rhizbmes were obtained by immersing the cores in distilled H20 and washing of?’the soil. One half of the washed roots and rhizomes of each core wcrc assayed at 0.05 atm OZ. and the other half at 0.20 atm 0,. The soil fraction was collected by crumbling the cores by hand and picking out as many roots as possible. All samples were ncubated at 24’ C. The rate of CzHz reduction was approximately hnear for the first 6 h of incubation. and then dropped somewhat for the final measurement at 9 h. Table I gives the rates for the first 6 h. From the results ofTable I. the CzHz reduction activity was about equally divided between the soil and root fractions. Since the soil fraction contained a substantial number offine roots, these may have been responsible for part of its activity. The sum of the activities of the root plus soil fractions was about one third that of the intact cores. This reduction in rate may have been due to loss of the soil immediate]] adjacent to the root surface, where N, fixation might be most active. Such soil was not present in the washed roots and may also have been removed from the soil fraction when the roots were taken out. The activity per gram of washed roots was about the same as reported hq Dabereiner it ul. (1972) for Purticurrl In(l.yiinl(ftl. The activit] was approximately the same at 0.05 and 0.20 atm pOZ. The results ofTable I must be considered with caution, since the soil cores were taken on 27 Scp 1973. about 3 days after autumn senescence. As mentioned pre\louslq, greater C,H, reduction rates were observed after senescence. so that it might be argued that the rhizosphere activity observed in Table I was due to N1 fixation associated with root decay. However, no root decay was apparent. 179
180
Short communication
,~tkIzoMiieri~c~rilr/its-The support, encouragement, and use of the laboratory facilities of Professor R. H. Burris are gratefuiiy acknowledged. This paper is Journal No. 87 of the University of Wisconsin (Madison) Arboretum. REFERENCES D~~IKKINFKJ.. DAY J. M. and DART P. J. (1972) Nitrogenase activity in the rhizosphere of sugar cant and some other tropical grasses. PI. Soil 37, 191-196.
DOMMERCZUES Y., BAL.ANVREAC’J.. RINALXX G., and WEINHARU P. (1973) Non-symbiotic nitrogen fixation in the rhizosphcre of rice, maize and different tropical grasses. Soil Rio/. Biochrln. 5, x3- x9. ROC~LKSC., MI-up I
Vol. 7, pp 179 to 180 Pergamon Press 1975 Prmted m Great Brnam.
SHORT COMMUNICATION Nitrogenase activity in the rhizosphere of Panicum uivptum J. TJEPIXMA Department
of Biochemistry.
University
Nitrogenase activity has been previously reported in the rhizosphere of Pmicm ~nazimmz, a tropical grass (Dcbereiner er al., 1972; Dommergues et al.. 1973). I here report nitrogenase activity in a temperate species of this genus, Pmicur~l uirgatum.
Soil cores (15 cm x 5.5 cm) were taken and placed in 13 x 29 cm Saran bags of 25 pm wall thickness. Saran was used because of its very low permeability to gases (Rogers et (II., 1956). The bags were closed by twisting the ends, folding them over, and wrapping with a rubber band. Nitrogenase activity was measured by the C,H, reduction method. Gases were added to and removed from the bags by puncturing them at a point reinforced by adhesive plastic tape. The puncture was sealed by additional tape. The assays were made in air with CZH, (generated from CaC:) added to approximately 0.1 atm. Gas samples were taken at intervals with 2.5 ml disposable syringes and were stored until analyzed some hours later. The soil cores were replaced in the ground during the incubation. The gas volume in the bags was determined by injecting a known volume of CZH, or CH, into the bags at the end of the experiment. The soil cores were taken from a stand of P. cirgururc on the Curtis Prairie, a reestablished prairie at the University of Wisconsin Arboretum. Measurements of C,H, were taken at 30 and 90 min after CZHz addition, and the difference in concentration was used in calculating the rate of
Table
I. CZH, reduction
by components
Soil core
nmol C?H,; core h- ’
A B c A B C D E F D E F G H I
2.1 5.6 2.8 2.5 18.5 5.1 7.5 6.0 4.2 6.9 7.9 6.9 37 75 50
Roots and rhizomes 0.05 atm 0, Roots and rhizomes 0.20 atm O2 Soil 0.05 atm Oz Soil 020 atm 0: Intact cores Air
of soil cores Fresh wt. per core (8) 21.3 18.4 IO.7 21.3 18.4 IO.7 468 508 525 468 50x 525 613 697 665
of Wisconsin,
Madison.
WI 53706, U.S.A
CZH2 reduction. The rates were linear with time, at lcast up to 3 h after CzHz addition. the longest time measured. Yellowing of the leaves due to autumn senescence occurred during the course of the measurements. The rate of CLHz reduction about 3. 5 and 6 days before senescence ranged between IO and 16 nmol CLHZ;core h- ‘. while about 5 days after senescence the activity ranged between 28 and 32 nmol C2Hz;lcorc h- ‘. Controls ( -CIHL) showed no measurable evolution of CzH4. The rates of CzHz reduction observed arc quite small when extrapolated on a seasonal and area basis. The rates observed before senescence correspond to about I.3 kg N; ha 120 days-‘. But it is quite possible that the rates are higher earlier in the growing season. The experiment of Table I was done to dctermme whether the observed CLHz reduction was associated with the roots of P. rirgutum. The intact cores were incubated in air as above, but the other measurements were done in 123 ml bottles in mixtures of Ar and 0:. with 0.1 atm C,H,. Washed roots and rhizbmes were obtained by immersing the cores in distilled H20 and washing of?’the soil. One half of the washed roots and rhizomes of each core wcrc assayed at 0.05 atm OZ. and the other half at 0.20 atm 0,. The soil fraction was collected by crumbling the cores by hand and picking out as many roots as possible. All samples were ncubated at 24’ C. The rate of CzHz reduction was approximately hnear for the first 6 h of incubation. and then dropped somewhat for the final measurement at 9 h. Table I gives the rates for the first 6 h. From the results ofTable I. the CzHz reduction activity was about equally divided between the soil and root fractions. Since the soil fraction contained a substantial number offine roots, these may have been responsible for part of its activity. The sum of the activities of the root plus soil fractions was about one third that of the intact cores. This reduction in rate may have been due to loss of the soil immediate]] adjacent to the root surface, where N, fixation might be most active. Such soil was not present in the washed roots and may also have been removed from the soil fraction when the roots were taken out. The activity per gram of washed roots was about the same as reported hq Dabereiner it ul. (1972) for Purticurrl In(l.yiinl(ftl. The activit] was approximately the same at 0.05 and 0.20 atm pOZ. The results ofTable I must be considered with caution, since the soil cores were taken on 27 Scp 1973. about 3 days after autumn senescence. As mentioned pre\louslq, greater C,H, reduction rates were observed after senescence. so that it might be argued that the rhizosphere activity observed in Table I was due to N1 fixation associated with root decay. However, no root decay was apparent. 179
180
Short communication
,~tkIzoMiieri~c~rilr/its-The support, encouragement, and use of the laboratory facilities of Professor R. H. Burris are gratefuiiy acknowledged. This paper is Journal No. 87 of the University of Wisconsin (Madison) Arboretum. REFERENCES D~~IKKINFKJ.. DAY J. M. and DART P. J. (1972) Nitrogenase activity in the rhizosphere of sugar cant and some other tropical grasses. PI. Soil 37, 191-196.
DOMMERCZUES Y., BAL.ANVREAC’J.. RINALXX G., and WEINHARU P. (1973) Non-symbiotic nitrogen fixation in the rhizosphcre of rice, maize and different tropical grasses. Soil Rio/. Biochrln. 5, x3- x9. ROC~LKSC., MI-up I