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II. Changes in Enzymes of Nitrogen Metabolism by VA Mycorrhizae in Ziziphus nummularia
J. P14nt PhysioJ.
Vol. 147. pp. 331-333 (1995)
II. Changes in Enzymes of Nitrogen Metabolism by VA Mycorrhizae in Ziziphus nummularia NISHI MATHUR
and ANIL VYAS
Department of Botany, J.N.V. University, P. Box 32, Jodhpur (Raj.) 342001lndia Received March 13, 1995 . Accepted June 28, 1995
Summary
Changes in enzymes of nitrogen metabolism by five VA mycorrhizae were investigated under glasshouse conditions in the arid fruit plant Ziziphus nummu/aria. The VA mycorrhizal plants had increased activities of GS, NR and GDH besides increasing protein accumulation in Z. nummu/aria. Among the five VAM species used during the present study, Glomus fascicu/atum (Thaxt. Sensu Gerd.) Gerd. and Trappe proved to be the most efficient VAM species. The findings can also be useful for producing highly proteinaceous leafy fodder of Z. nummu/aria. Details about the symbiotic relationship are also discussed.
Key words: Enzymes, nitrogen metabolism, root colonization, VAAf, Ziziphus nummu/aria. Abbreviations: GDH ductase.
=
Glutamine dehydrogenase; GS
Introduction
Nitrogen is the most important mineral nutrient for which availability is most likely limiting to plant growth (McArthur and Knowles, 1993). Due to its beneficial effects VA mycorrhizae are receiving considerable attention in agriculture and forestry (Peterson et al., 1984). Ziziphus nummu/aria is an important multipurpose fruit plant of arid and semi-arid regions. It is a source of fuel, fodder and timber. Cultivation of the plant is done mostly in p-deficient sandy soils of drought prone areas. The primary stresses imposed on vegetation by arid environment are lack of water and mineral nutrients (Fisher and Turner, 1978). The VAM fungi may be of particular significance in coping with p-deficiency stress in natural ecosystems (Eissenstat et al., 1993). Improved biomass production and nutrient uptake by VA mycorrhizae in Z. nummu/aria has been reported (Mathur and Vyas, 1994). There has been less work on nitrogen metabolism in plants colonized by VAM (Cliquet and Stewart, 1.993). Keeping all these facts in mind the present investigation was undertaken to evaluate the efficiency of different V.AM fungi on activities of enzymes of nitrogen metabolism, VIZ. GS, NR and GDH in Z. nummu/aria. © 1995 by Gustav Fischer Vedag, Stuttgart
=
Glutamine synthetase; NR
nitrate re-
Materials and Methods
Five VAM species, namely Glomus fascicu14tum (Than. sensu Gerd) Gerd. and Trappe, G. mosseae (Nicol. and Gerd.) Gerd. and Trappe, Gigaspora margarita Becker and Hall, Sclerocystis rumformis Gerd. and Trappe, and Scutellospora calospora (Nicol. and Gerd.) Walker and Sanders collected from rhizosphere soil of Ziziphus nummu14ria were maintained on Cenchrus ciliaris to prepare pot cultures. One the pot cultures were established, the soil from these pot cultures along with roots of C ciliaris were used as source of inoculum. The mycorrhizal plants received 10 g inoculum in each pot of 18 cm diameter containing sterilized soil (Mathur and Vyas, 1995), while non-mycorrhizal plants did not receive any treatment. The surface sterilized seeds of Ziziphus nummularia were sown and kept under glasshouse conditions. The seedlings were thinned to one per pot after 15 days. There were twenty replicates for each treatment. Plants were grown in a glasshouse having 50 % humidity with a 16-h day length at 24-26 ae. The nutrient solution used was 20 % Long Ashton enriched with phosphorus and nitrogen according to the procedure of Koide and Li (1990). Roots collected from the plant materials were suitably processed and stained (Phillips and Hayman, 1970), and percentage of root colonization was calculated by the Grid line intersect method (Giovannetti and Mosse, 1980). For all studies except that on NR, 6O-day-old plants were harvested, and the roots and shoots were separated, weighed, immersed in
332
NISHI MATHUR and ANIL VYAS
liquid nitrogen, stored at - 80°C, and analyzed withi~ 4~ h..Plant tissue, O.5g fresh weight of each organ, was ground In .lIquld N z and extracted in 5mL of buffer. The GS/GDH extractIOn buffer (pH 8.0) contained 25 rnM Tris, 1mM EDTA, 1mM DTT, 1mM reduced GSH, 10 mM MgS0 4, 5 mM Glu, 1 % PVP and 0.5 % Nonidet P-40. After centrifugation, the supernatants were used for . enzyme and soluble protein assays as follows. GS (EC 6.3.1.2) was determined by the transferase assay (Snaplro and Stadtman, 1970). The reaction mixture contained 80J.1mol Mes, 60 J.1mol L-Gln, 25 J.1mol NazAs0 4, 2.5 J.1mol hydroxylamine, 2 J.1mol MnCh and 15 J.1mol ADP (Final pH 7.6). The reaction was initiated by addition of 0.10 mL of enzyme extract and terminated after 10 min for shoots and 20 min for roots by addition of 1mL of ferric chloride reagent. After the mixture was centrifuged, A500 was dertermined. GDH (EC 2.6.1.53) was assayed by determining the rate of 2-oxoglutarate NADH or NADPH oxidation. The reaction mixture contained 150 J.1mol NH 4C1, 1 J.1mol CaCh, 0.3 mol NAD (P) H, 20 J.1mol 2-oxoglutarate, and 100 J.1mol Tris buffer (final pH 8.2 and final volume 1mL). NR activity was measured by an in vivo assay (Stewart and Orebamjo, 1979) from small pieces of roots o~ shoots: All enzyme assays were carried out at 30°C and were linear With respect to length of incubation time and quantity of enzyme assayed. Soluble protein was determined by the comassie blue method (Bradford, 1976) in roots and leaves.
Results and Discussion
The buffer soluble protein concentration was very low in roots (between 0.80-1.97 gkg- I fresh weight) while it was higher in concentration in shoots (Table 1). VAM colonized plants showed higher accumulation of protein than nonmycorrhizal ones. Different VAM species varied in their efficacy to increase protein concentration in both of the organs. Among the five VAM species used during the present investigation G. fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe increased the soluble protein concentration in both of the organs most efficiently, while Sclerocystis rubiformis Gerd. and Trappe responded least effectively. This increase in soluble protein concentration was similar to the earlier findings of VAM inoculated Zea mays 1. (Cliquet and Stewart, 1993). Nitrate reductase activity was in the range of 0.14 to 0.321J.mol of nitrate produced h- I g-I fresh weight in roots Table 1: Influence of VA mycorrhizae on soluble protein, glutamine synthetase and nitrate reductase activity of Ziziphus num· mularia. Treatment
Control G. fascia/latum G. mosseae Gig. margarita Scle. rubiformis Scu. calospora L.S.D. at P :s 0.05
Soluble protein gkg- I fresh weight
GS NR mmol h- I kg-I fresh weight
Root
Leaves
Root
Shoot
Root
Shoot
0.80 1.97 1.78 1.56 1.12 1.40 0.23
7.10 14.12 13.14 12.20 9.21 11.72 2.13
56.10 112.16 95.32 90.15 74.06 82.17 0.13
57.24 126.40 115.12 98.42 79.32 89.50 0.14
0.14 0.32 0.27 0.25 0.24 0.23 0.02
0.21 0.43 0.34 0.32 0.25 0.29 0.02
and 0.21 to 0.431J.mol nitrate produced h- I g-I fresh weight in shoots. Activity of this enzyme was also found to be higher in both organs in all of the VAM plants compared with the control. Similar enhancement of nitrate reductase activity was reported in roots and leaves of VAM infected clover and was attributed to improved p-nutrition provided by VAM symbiosis (Oliver et al., 1983). G. fasciculatum reo sulted in a more than two-fold increase in NR activity in shoots and roots of Z. nummularia. McArthur and Knowles (1993) observed a 20% increased NR activity in VAM inoculated potato plants. Endomycorrhizal fungal species like G. macrocarpum and G. mosseae have also been known to reduce nitrate ions (Ho and Trappe, 1975). The results in the present investigation suggest that with a capacity for reducing nitrate it is likely that the symbiotic effectiveness of the VAM fungi is enhanced in terms of nitrogen assimilation and translocation to the host plant. GS activity was twice as high in G. fasciculatum inoculated Z. nummularia plants compared with control plants in both roots (Table 1, from 56.10-112.16IJ.mol of hydroxamate produced h-Ig- I fresh weight) and shoots (from 57.24126.40 IJ.mol of hydroxamate produced h- I g-I fresh weight). Smith et al. (1985) showed by enzymatically separating fungal and plant tissues that G. mosseae contribute directly to GS activity in onion roots. In VAM roots higher GS activity may be accompanied by increased amino acid synthesis and an increase in 15 N-Gln translocation through the xylem. Cliquet and Stewart (1993) reported increased ammonium assimilation, GIn production and xylen nitrogen translocation in VA mycorrhizal maize plants. NADPH dependent GDH activity was increased by VAM fungal colonization but remained quite low compared with NADH dependent activity. VAM inoculation resulted in efficiently increasing the GDH activity in roots as compared with shoots (Table 2). G. fasciculatum inoculation resulted in a more than three-fold increase in root GDH activity during the present study. The presence of NADP.GDH, characteristic of fungi (Sims and Folkes, 1964), indicated that colonized Z. nummularia roots have the potential for nitrogen metabolism through the fungal pathway. These Z. num· mularia roots differ from those of onion and clover (Smith et al., 1985) in having high levels of NAD-GDH. The present investigation reveals that different VAM fu~gi vary in their response to increase GS, NR and GDH actIv-
Table 2: Influence of VA mycorrhizae on glutamine dehydrogenase activity of Ziziphus nummularia. Treatment
Control G. fasciculatum G. mosseae Gig. margarita Scle. rubiformis Scu. calospora L.S.D. at P s 0.10
NADH-GDH NADPH-GDH mmolh-Ikg- I fresh weight Root
Shoot
Root
Shoot
0.24 0.78 0.55 0.47 0.34 0.39 0.10
1.10 1.29 1.22 1.19 1.13 1.16 0.04
0.19 0.65 0.55 0.49 0.32 0.40 0.07
1.03 1.21 1.18 1.15 1.08 1.12 0.02
Nitrogen metabolism and VA mycorrhizae Table 3: Spore population and percentage of root colonization by VA mycorrhizae in rhizosphere of Ziziphus nummularia. Treatment
VAM spores 100g- 1 soil
Percentage root colonization
590 445 365 210 295
85 75 64 50 55
Control
G. fasciculatum G. mosseae Gig. margarita Scle. rubiformis Scu. calospora
ities, as well as the soluble protein concentration. However, a maximal increase in activities of all these enzymes and protein levels was observed in G. fasciculatum inoculated plants, which further confirms the efficiency of this VAM strain to be most suitable for Z. nummularia as this strain also increased activities of PRO and PPO significantly in this fruit plant (Mathur and Vyas, 1995). This capacity of G. fasciculatum might be due to efficient root colonization by this VAM endophyte (Table 3). Hence, it can be concluded that G. fasciculatum can be used as a most efficient VAM species for Z. nummularia as this VAM endophyte increased various enzymatic activities of nitrogen metabolism most effectively. The colonized roots of Z. nummularia have the potential for nitrogen metabolism through the fungal pathway. The findings can also be useful in producing highly proteinaceous leafy fodder of Z. nummularia as the G. fasciculatum increased (more than two-fold) the protein contents in leaves of this multipurpose fruit plant of arid and semi-arid regions. Acknowledgement
The Senior author is thankful to C.S.I.R. New Delhi for financial support.
References BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248-254 (1976). CUQUET, J. B. and G. R. STEWART: Ammonia assimilation in Zea mays L. infected with a vesicular arbuscular mycorrhizal fungus Glomusfasciculatum. Plant Physiol. 101, 865-871 (1993).
333
EISSENSTAT, D. M., J. H. GRAHAM, J. P. SYVERTSEN, and D. L. DROUILLAR: Carbon economy of sour orange in relation to mycorrhizal colonization and phosphours status. Ann. Bot. 71, 1-10 (1993). FISHER, R. A. and N. C. TURNER: Plant productivity in the arid and semi-arid zones. Ann. Rev. PI. Physiol. 29, 277 -317 (1978). GIOVANNETII, M. and B. MOSSE: An evaluation of techniques for measuring VAM infection in roots. New Phytol. 84, 489-500 (1980). Ho, I. and J. M. TRAPPE: Nitrate reductase capacity of two vesicular arbuscular mycorrhizal fungi. Mycologia. 4, 886 (1975). KOIDE, R. T. and M. LI: One host regulation of the vesicular arbuscular mycorrhizal symbiosis. New Phytol. 114, 59-74 (1990). MATHUR, N. and A. VYAs: Relative efficiency of different VAM fungi on biomass production, nutrient uptake and physiology of Ziziphus nummularia. Indian Forester (1994). (In press). - - Changes in isozyme patterns of peroxidase and polyphenol oxidase by VAM fungi in roots of Ziziphus species. J. Plant Physiol. 145, 498-500 (1995). McARTHUR, D. A. J. and N. R. KNOWLES: Influence of vesicular arbuscular mycorrhizal fungi on the response of potato to phosphorus deficiency. Plant Physiol. 101, 147 -160 (1993). OLIVER, A. J., S. SMITH, D. J. D. NICHOLAS, W. WALLECE, and F. A. SMITH: Activities of nitrate reductase in Trifolium subterranium L.: effect of mycorrhizal infection and phosphate nutrition. New Phytol. 94, 63-79 (1983). PETERSON, R. L., Y. PICHE, and C. PLENCHETIE: Mycorrhizae and their potential use in agricultural and forestry industries. Biotech. Adv. 2, 101-120 (1984). PHILLIPS, J. M. and D. S. HAYMAN: Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158-160 (1970). SHAPIRO, B. M. and E. R. STADTMAN: Glutamine synthetase (Escherichia coli): Methods Enzymol. 17 A, 910-922 (1970). SIMS, A. P. and B. F. FOLKES: A kinetic study of the assimilation of Nammodia and synthesis of amino acids in an exponentially growing culture of Candida utilis. Proc. R. Soc. BioI. 159, 479502 (1964). SMITH, S. E., B. J. ST. JOHN, F. A. SMITH, and D. J. D. NICHOLAS: Activity of glutamine synthetase and glutamate dehydrogenase in Allium cepa L. and Trifolium subterraneum L.: effect of mycorrhizal infection and phosphate nutrition. New Phytol. 99, 211227 (1985). STEWART, G. R. and T. OREBAMJO: Some unusual characteristics of nitrate reduction in Erythrina Senegalensis. New Phyol. 83, 311-319 (1979).
Vol. 147. pp. 331-333 (1995)
II. Changes in Enzymes of Nitrogen Metabolism by VA Mycorrhizae in Ziziphus nummularia NISHI MATHUR
and ANIL VYAS
Department of Botany, J.N.V. University, P. Box 32, Jodhpur (Raj.) 342001lndia Received March 13, 1995 . Accepted June 28, 1995
Summary
Changes in enzymes of nitrogen metabolism by five VA mycorrhizae were investigated under glasshouse conditions in the arid fruit plant Ziziphus nummu/aria. The VA mycorrhizal plants had increased activities of GS, NR and GDH besides increasing protein accumulation in Z. nummu/aria. Among the five VAM species used during the present study, Glomus fascicu/atum (Thaxt. Sensu Gerd.) Gerd. and Trappe proved to be the most efficient VAM species. The findings can also be useful for producing highly proteinaceous leafy fodder of Z. nummu/aria. Details about the symbiotic relationship are also discussed.
Key words: Enzymes, nitrogen metabolism, root colonization, VAAf, Ziziphus nummu/aria. Abbreviations: GDH ductase.
=
Glutamine dehydrogenase; GS
Introduction
Nitrogen is the most important mineral nutrient for which availability is most likely limiting to plant growth (McArthur and Knowles, 1993). Due to its beneficial effects VA mycorrhizae are receiving considerable attention in agriculture and forestry (Peterson et al., 1984). Ziziphus nummu/aria is an important multipurpose fruit plant of arid and semi-arid regions. It is a source of fuel, fodder and timber. Cultivation of the plant is done mostly in p-deficient sandy soils of drought prone areas. The primary stresses imposed on vegetation by arid environment are lack of water and mineral nutrients (Fisher and Turner, 1978). The VAM fungi may be of particular significance in coping with p-deficiency stress in natural ecosystems (Eissenstat et al., 1993). Improved biomass production and nutrient uptake by VA mycorrhizae in Z. nummu/aria has been reported (Mathur and Vyas, 1994). There has been less work on nitrogen metabolism in plants colonized by VAM (Cliquet and Stewart, 1.993). Keeping all these facts in mind the present investigation was undertaken to evaluate the efficiency of different V.AM fungi on activities of enzymes of nitrogen metabolism, VIZ. GS, NR and GDH in Z. nummu/aria. © 1995 by Gustav Fischer Vedag, Stuttgart
=
Glutamine synthetase; NR
nitrate re-
Materials and Methods
Five VAM species, namely Glomus fascicu14tum (Than. sensu Gerd) Gerd. and Trappe, G. mosseae (Nicol. and Gerd.) Gerd. and Trappe, Gigaspora margarita Becker and Hall, Sclerocystis rumformis Gerd. and Trappe, and Scutellospora calospora (Nicol. and Gerd.) Walker and Sanders collected from rhizosphere soil of Ziziphus nummu14ria were maintained on Cenchrus ciliaris to prepare pot cultures. One the pot cultures were established, the soil from these pot cultures along with roots of C ciliaris were used as source of inoculum. The mycorrhizal plants received 10 g inoculum in each pot of 18 cm diameter containing sterilized soil (Mathur and Vyas, 1995), while non-mycorrhizal plants did not receive any treatment. The surface sterilized seeds of Ziziphus nummularia were sown and kept under glasshouse conditions. The seedlings were thinned to one per pot after 15 days. There were twenty replicates for each treatment. Plants were grown in a glasshouse having 50 % humidity with a 16-h day length at 24-26 ae. The nutrient solution used was 20 % Long Ashton enriched with phosphorus and nitrogen according to the procedure of Koide and Li (1990). Roots collected from the plant materials were suitably processed and stained (Phillips and Hayman, 1970), and percentage of root colonization was calculated by the Grid line intersect method (Giovannetti and Mosse, 1980). For all studies except that on NR, 6O-day-old plants were harvested, and the roots and shoots were separated, weighed, immersed in
332
NISHI MATHUR and ANIL VYAS
liquid nitrogen, stored at - 80°C, and analyzed withi~ 4~ h..Plant tissue, O.5g fresh weight of each organ, was ground In .lIquld N z and extracted in 5mL of buffer. The GS/GDH extractIOn buffer (pH 8.0) contained 25 rnM Tris, 1mM EDTA, 1mM DTT, 1mM reduced GSH, 10 mM MgS0 4, 5 mM Glu, 1 % PVP and 0.5 % Nonidet P-40. After centrifugation, the supernatants were used for . enzyme and soluble protein assays as follows. GS (EC 6.3.1.2) was determined by the transferase assay (Snaplro and Stadtman, 1970). The reaction mixture contained 80J.1mol Mes, 60 J.1mol L-Gln, 25 J.1mol NazAs0 4, 2.5 J.1mol hydroxylamine, 2 J.1mol MnCh and 15 J.1mol ADP (Final pH 7.6). The reaction was initiated by addition of 0.10 mL of enzyme extract and terminated after 10 min for shoots and 20 min for roots by addition of 1mL of ferric chloride reagent. After the mixture was centrifuged, A500 was dertermined. GDH (EC 2.6.1.53) was assayed by determining the rate of 2-oxoglutarate NADH or NADPH oxidation. The reaction mixture contained 150 J.1mol NH 4C1, 1 J.1mol CaCh, 0.3 mol NAD (P) H, 20 J.1mol 2-oxoglutarate, and 100 J.1mol Tris buffer (final pH 8.2 and final volume 1mL). NR activity was measured by an in vivo assay (Stewart and Orebamjo, 1979) from small pieces of roots o~ shoots: All enzyme assays were carried out at 30°C and were linear With respect to length of incubation time and quantity of enzyme assayed. Soluble protein was determined by the comassie blue method (Bradford, 1976) in roots and leaves.
Results and Discussion
The buffer soluble protein concentration was very low in roots (between 0.80-1.97 gkg- I fresh weight) while it was higher in concentration in shoots (Table 1). VAM colonized plants showed higher accumulation of protein than nonmycorrhizal ones. Different VAM species varied in their efficacy to increase protein concentration in both of the organs. Among the five VAM species used during the present investigation G. fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe increased the soluble protein concentration in both of the organs most efficiently, while Sclerocystis rubiformis Gerd. and Trappe responded least effectively. This increase in soluble protein concentration was similar to the earlier findings of VAM inoculated Zea mays 1. (Cliquet and Stewart, 1993). Nitrate reductase activity was in the range of 0.14 to 0.321J.mol of nitrate produced h- I g-I fresh weight in roots Table 1: Influence of VA mycorrhizae on soluble protein, glutamine synthetase and nitrate reductase activity of Ziziphus num· mularia. Treatment
Control G. fascia/latum G. mosseae Gig. margarita Scle. rubiformis Scu. calospora L.S.D. at P :s 0.05
Soluble protein gkg- I fresh weight
GS NR mmol h- I kg-I fresh weight
Root
Leaves
Root
Shoot
Root
Shoot
0.80 1.97 1.78 1.56 1.12 1.40 0.23
7.10 14.12 13.14 12.20 9.21 11.72 2.13
56.10 112.16 95.32 90.15 74.06 82.17 0.13
57.24 126.40 115.12 98.42 79.32 89.50 0.14
0.14 0.32 0.27 0.25 0.24 0.23 0.02
0.21 0.43 0.34 0.32 0.25 0.29 0.02
and 0.21 to 0.431J.mol nitrate produced h- I g-I fresh weight in shoots. Activity of this enzyme was also found to be higher in both organs in all of the VAM plants compared with the control. Similar enhancement of nitrate reductase activity was reported in roots and leaves of VAM infected clover and was attributed to improved p-nutrition provided by VAM symbiosis (Oliver et al., 1983). G. fasciculatum reo sulted in a more than two-fold increase in NR activity in shoots and roots of Z. nummularia. McArthur and Knowles (1993) observed a 20% increased NR activity in VAM inoculated potato plants. Endomycorrhizal fungal species like G. macrocarpum and G. mosseae have also been known to reduce nitrate ions (Ho and Trappe, 1975). The results in the present investigation suggest that with a capacity for reducing nitrate it is likely that the symbiotic effectiveness of the VAM fungi is enhanced in terms of nitrogen assimilation and translocation to the host plant. GS activity was twice as high in G. fasciculatum inoculated Z. nummularia plants compared with control plants in both roots (Table 1, from 56.10-112.16IJ.mol of hydroxamate produced h-Ig- I fresh weight) and shoots (from 57.24126.40 IJ.mol of hydroxamate produced h- I g-I fresh weight). Smith et al. (1985) showed by enzymatically separating fungal and plant tissues that G. mosseae contribute directly to GS activity in onion roots. In VAM roots higher GS activity may be accompanied by increased amino acid synthesis and an increase in 15 N-Gln translocation through the xylem. Cliquet and Stewart (1993) reported increased ammonium assimilation, GIn production and xylen nitrogen translocation in VA mycorrhizal maize plants. NADPH dependent GDH activity was increased by VAM fungal colonization but remained quite low compared with NADH dependent activity. VAM inoculation resulted in efficiently increasing the GDH activity in roots as compared with shoots (Table 2). G. fasciculatum inoculation resulted in a more than three-fold increase in root GDH activity during the present study. The presence of NADP.GDH, characteristic of fungi (Sims and Folkes, 1964), indicated that colonized Z. nummularia roots have the potential for nitrogen metabolism through the fungal pathway. These Z. num· mularia roots differ from those of onion and clover (Smith et al., 1985) in having high levels of NAD-GDH. The present investigation reveals that different VAM fu~gi vary in their response to increase GS, NR and GDH actIv-
Table 2: Influence of VA mycorrhizae on glutamine dehydrogenase activity of Ziziphus nummularia. Treatment
Control G. fasciculatum G. mosseae Gig. margarita Scle. rubiformis Scu. calospora L.S.D. at P s 0.10
NADH-GDH NADPH-GDH mmolh-Ikg- I fresh weight Root
Shoot
Root
Shoot
0.24 0.78 0.55 0.47 0.34 0.39 0.10
1.10 1.29 1.22 1.19 1.13 1.16 0.04
0.19 0.65 0.55 0.49 0.32 0.40 0.07
1.03 1.21 1.18 1.15 1.08 1.12 0.02
Nitrogen metabolism and VA mycorrhizae Table 3: Spore population and percentage of root colonization by VA mycorrhizae in rhizosphere of Ziziphus nummularia. Treatment
VAM spores 100g- 1 soil
Percentage root colonization
590 445 365 210 295
85 75 64 50 55
Control
G. fasciculatum G. mosseae Gig. margarita Scle. rubiformis Scu. calospora
ities, as well as the soluble protein concentration. However, a maximal increase in activities of all these enzymes and protein levels was observed in G. fasciculatum inoculated plants, which further confirms the efficiency of this VAM strain to be most suitable for Z. nummularia as this strain also increased activities of PRO and PPO significantly in this fruit plant (Mathur and Vyas, 1995). This capacity of G. fasciculatum might be due to efficient root colonization by this VAM endophyte (Table 3). Hence, it can be concluded that G. fasciculatum can be used as a most efficient VAM species for Z. nummularia as this VAM endophyte increased various enzymatic activities of nitrogen metabolism most effectively. The colonized roots of Z. nummularia have the potential for nitrogen metabolism through the fungal pathway. The findings can also be useful in producing highly proteinaceous leafy fodder of Z. nummularia as the G. fasciculatum increased (more than two-fold) the protein contents in leaves of this multipurpose fruit plant of arid and semi-arid regions. Acknowledgement
The Senior author is thankful to C.S.I.R. New Delhi for financial support.
References BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248-254 (1976). CUQUET, J. B. and G. R. STEWART: Ammonia assimilation in Zea mays L. infected with a vesicular arbuscular mycorrhizal fungus Glomusfasciculatum. Plant Physiol. 101, 865-871 (1993).
333
EISSENSTAT, D. M., J. H. GRAHAM, J. P. SYVERTSEN, and D. L. DROUILLAR: Carbon economy of sour orange in relation to mycorrhizal colonization and phosphours status. Ann. Bot. 71, 1-10 (1993). FISHER, R. A. and N. C. TURNER: Plant productivity in the arid and semi-arid zones. Ann. Rev. PI. Physiol. 29, 277 -317 (1978). GIOVANNETII, M. and B. MOSSE: An evaluation of techniques for measuring VAM infection in roots. New Phytol. 84, 489-500 (1980). Ho, I. and J. M. TRAPPE: Nitrate reductase capacity of two vesicular arbuscular mycorrhizal fungi. Mycologia. 4, 886 (1975). KOIDE, R. T. and M. LI: One host regulation of the vesicular arbuscular mycorrhizal symbiosis. New Phytol. 114, 59-74 (1990). MATHUR, N. and A. VYAs: Relative efficiency of different VAM fungi on biomass production, nutrient uptake and physiology of Ziziphus nummularia. Indian Forester (1994). (In press). - - Changes in isozyme patterns of peroxidase and polyphenol oxidase by VAM fungi in roots of Ziziphus species. J. Plant Physiol. 145, 498-500 (1995). McARTHUR, D. A. J. and N. R. KNOWLES: Influence of vesicular arbuscular mycorrhizal fungi on the response of potato to phosphorus deficiency. Plant Physiol. 101, 147 -160 (1993). OLIVER, A. J., S. SMITH, D. J. D. NICHOLAS, W. WALLECE, and F. A. SMITH: Activities of nitrate reductase in Trifolium subterranium L.: effect of mycorrhizal infection and phosphate nutrition. New Phytol. 94, 63-79 (1983). PETERSON, R. L., Y. PICHE, and C. PLENCHETIE: Mycorrhizae and their potential use in agricultural and forestry industries. Biotech. Adv. 2, 101-120 (1984). PHILLIPS, J. M. and D. S. HAYMAN: Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158-160 (1970). SHAPIRO, B. M. and E. R. STADTMAN: Glutamine synthetase (Escherichia coli): Methods Enzymol. 17 A, 910-922 (1970). SIMS, A. P. and B. F. FOLKES: A kinetic study of the assimilation of Nammodia and synthesis of amino acids in an exponentially growing culture of Candida utilis. Proc. R. Soc. BioI. 159, 479502 (1964). SMITH, S. E., B. J. ST. JOHN, F. A. SMITH, and D. J. D. NICHOLAS: Activity of glutamine synthetase and glutamate dehydrogenase in Allium cepa L. and Trifolium subterraneum L.: effect of mycorrhizal infection and phosphate nutrition. New Phytol. 99, 211227 (1985). STEWART, G. R. and T. OREBAMJO: Some unusual characteristics of nitrate reduction in Erythrina Senegalensis. New Phyol. 83, 311-319 (1979).