Bot. Bull. Acad. Sin. (1996) 37: 209-212

Mathur and Vyas — Biochemical changes in Ziziphus xylopyrus by VA mycorrhizae

Biochemical changes in Ziziphus xylopyrus by VA mycorrhizae

Nishi Mathur¹ and Anil Vyas

Post Box No. 32, Department of Botany, Jai Narain Vyas University, Jodhpur- 342 001, (Raj.) India

(Received January 4, 1995; Accepted March 15, 1996)

Abstract. The efficacy of six VAM species, Acaulospora morrowae Spain & Schenck, Gigaspora margarita Becker & Hall, Glomus fasciculatum (Thaxt. Sensu. Gerd.) Gerd. & Trappe, G. macrocarpum Tul. & Tul. Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders, and Sclerocystis rubiformis Gerd. & Trappe, collected from rhizosphere soils of Ziziphus xylopyrus, was evaluated for enhancement of NR, GS, GDH, PRO, PPO activities and protein, phenolic, and catechin contents in this fruit tree. Culturing was done under glass house conditions and analyses were performed 180 days after inoculation. All fungi showed beneficial effects, with S. calospora being the most promoting of all biochemical parameters.

Keywords: Glutamine dehydrogenase; Glutamine synthetase; Nitrate reductase; Peroxidase; Polyphenol oxidase; Mycorrhizae; VAM; Ziziphus xylopyrus.

Abbreviations: GDH, glutamine dyhydrogenase; GS, glutamine synthetase; NR, nitrate reductase; PRO, peroxidase; PPO, polyphenol oxidase; VAM, vesicular arbuscular mycorrhizae.

Introduction

Ziziphus xylopyrus is an important multipurpose fruit tree of the arid and semi arid regions of Asia. It is a source of fuel, fodder, and timber, besides producing the nutritious fruit known as ber. VAM fungi are well known to bring about physiological changes in plants by increasing various enzymatic activities (Mathur and Vyas, 1995). Nitrogen is the nutrient, of which availability is most likely limiting to plant growth (Mc Arthur and Knowles, 1993). Increased nitrogen uptake by VA mycorrhizae has been well recognized (Cliquet and Stewart, 1993). Nitrate reductase, glutamine synthetase, and glutamine dehydrogenase are important enzymes of nitrogen metabolism. Peroxidase and polyphenol oxidase are important components of the defence mechanism of plants against pathogens. Phenols and catechins are also important in plant disease resistance. (Protein levels in leaves determine the nutritive value of fodder). In view of these benefits, this investigation was undertaken to evaluate the efficiency of different VAM species towards biochemical enhancement in Z. xylopyrus.

Materials and Methods

Six VAM species—Acaulospora morrowae Spain & Schenck, Gigaspora margarita Becker & Hall, Glomus fasciculatum (Thaxt. Sensu. Gerd.) Gerd. & Trappe, G. macrocarpum Tul. & Tul. Sclerocystis rubiformis Gerd. & Trappe, and Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders—were examined. Fungal samples were collected by wet sieving and decanting (Gerdemann and Nicolson, 1963) soil samples from the rhizosphere of

Ziziphus xylopyrus. The fungi were identified following the synoptic key of Trappe (1982) and the manual of Schenck and Perez (1987) and maintained in pot cultures of Cenchrus ciliaris. Soil samples from the pot cultures, along with infected root segments of C. ciliaris, were used as inoculum. Twenty g of inoculum containing about 5,000 spores and infected root segments were inoculated in each 18-cm diameter pot containing sterilized soil. The soil used was sandy, with a pH of 8.2, available p of 2.30 ppm, total N of 2.15 ppm, and organic carbon of 0.12 percent. Five surface sterilized seeds (sterilized by 0.1% Hgcl₂ solution) of Z. xylopyrus were sown per pot. The pots were placed in a glass house at 60 percent humidity, 22_25°C and 12_14 h day length. The pots without VAM inoculum served as control.

All pots were supplied with half strength Hoaglands' nutrient solution every 15 days. After two weeks seedlings were thinned to one per pot. There were ten pots per treatment. After six months the plants were harvested. Phosphorus was estimated by the vanadomolybdate method (Jackson, 1973). Soluble proteins in leaves and roots were determined by the comassive blue method (Bradford, 1976) and total phenols were determined by using Folin ciocalteu's reagent (Bray and Thrope, 1954). For estimation of catechins, root samples were steamed and dried to constant weight. Catechins extracted in 50 per cent acetone were estimated by measuring absorbance by the yellow colour that developed with diazotized sulphanilamide at 430 nm (Gulati et al., 1993).

GS, GDH and NR Assay

Plant tissue, 0.5 g fresh weight per organ, was ground in liquid N₂ and extracted in 5 ml buffer. The GS, GDH extraction buffer (pH 8.0) contained 25 mm Tris, 1 mm

¹Corresponding author.

Botanical Bulletin of Academia Sinica, Vol. 37, 1996

EDTA, 1 mm DDT, 1 mm reduced GSH, 10 nM MgSo₄, 5 nM Glu, 1% PVP, and 0.5% Nonidet p-40. After centrifugation, the supernatants were used for enzyme and soluble protein assays as follows: GS was determined by the transferase assay (Shapiro and Stadtman, 1970). The reaction mixture contained 80 mmol of Mes, 60 mmol of L- Gln, 25 mmol of Na₂ ASO₄, 2.5 mol of hydroxylamine, 2 mmol of Mn Cl₂, and 15 mmol of ADP (Final pH 7.6). The reaction was initiated by adding 0.10 ml of enzyme extract and terminated after 10 min for shoots and 20 min for roots by adding 1 ml of ferric chloride reagent. The mixture was contrifuged and A₅₀₀ was measured in the supernatant. NR activity was measured by the in vivo assay of Stewart and Orebamjo (1979) from small pieces of roots and shoots. GDH was assayed by determining the rate of 2-oxogluarate NADH oxidation. The reaction mixture contained 150 mmol of NH₄Cl, 1 mmol of CaCl₂, 0.3 mmol of NADPH, 20 mmol of 2 oxogluatrate and 100 mmol of Tris buffer (Final pH 8.2 and final volume 1 ml).

PRO and PPO Assay

Root pieces were homogenized in 0.1 M phosphate buffer, pH 7.0, with a pre-chilled mortar and pestle at 4°C. The homogenate was centrifuged at 5,000 g for 15 min and the supernatant was used for enzyme assay. Peroxidase activity was measured by incubating the enzyme with guaiacol and hydrogen peroxide (Racusen and Foote, 1965). The arbitrary unit of enzyme activity chosen was change in absorbance of 0.001 sec^-1. Polyphenol oxidase activity was measured at 420 nm, using the method of Mahadevan (1975). The activity is presented in terms of absorbance of 100 mg (fresh weight of tissues)^-1. At harvest time ten samples of each treatment were analyzed.

Results and Discussion

Observations regarding changes in NR, GS, and GDH activities are presented in Table 1. NR activity was in the range of 0.12_0.27 mmol nitrite produced h^-1 g^-1 fresh weight in roots and 0.17_0.35 mmol h^-1 g^-1 in shoots. Activity of this enzyme was higher in both organs in all VAM inoculated seedlings when compared with the control. However, the VAM species varied in their effects on this enzyme. Inoculated S. calospora plants showed a more

than twofold increase in NR activity. A similar enhancement of NR activity in roots and leaves of VAM infected clover was attributed to an improved P-nutrition (Oliver et al., 1983). The highest GS activity in roots and shoots of Z. xylopyrus was observed with S. calospora inoculated plants, and the smallest increase was observed in A. morrowae inoculated plants. Smith et al. (1985) reported that G. mosseae contributed positively to GS activity in onion roots. In VAM infected roots, higher GS activity is probably accompanied by increased amino acid synthesis. Cliquet and Stewart (1993) reported increased ammonia assimilation, Gln production and xylem nitrogen translocation in VA mycorrhiza infected maize plants. VA mycorrhizal inoculation increased NADP_GDH activity more prominently in roots than shoots. S. calospora inoculated plants showed a more than threefold increased of NADP_GDH activity in roots of Z. xylopyrus. Cliquet and Stewart (1993) also observed an increased NADP_GDH activity in VA mycorrhizal roots. The presence of NADP_GDH, characteristic of fungi (Sims and Folkes, 1964) indicates that VAM colonized Z. xylopyrus roots have the potential for nitrogen metabolism through the fungal pathway.

Whatever the mechanism, the three key enzymes of inorganic nitrogen assimilation in Z. xylopyrus are enhanced most effectively by S. calospora. VAM inoculation also increased peroxidase and polyphenoloxidase activities in roots of Z. xylopyrus (Table 2). The increased peroxidase activity by VAM fungi may be due to an increased P-uptake resultant from the symbiosis. McArthur and Knowles (1992) reported lower peroxidase activity in low P roots than high P roots.

A positive correlation was observed between total phenol accumulation and PPO activity in VAM inoculated Z. xylopyrus roots (Table 2). S. calospora also was most efficient in enhancing phenolic accumulation. The increased phenolic accumulation might have resulted from an increased PPO activity. Accumulation of phenol in VA mycorrhizal plants has been reported (Krishna and Bagyaraj, 1984). as well as in tissues of a variety of plants during pathogenesis (Vidhyasekaran, 1989). PRO and PPO are important in the defence mechanism against pathogens. Both enzymes are involved in the oxidation of phenolic compounds to quinones, which are presumably toxic to

Table 1. Changes in GS, NR and GDH activities in Z. xylopyrus resultant from infection by different VAM fungi.

Treatment Soluble protein GS NR NADPH-GDH

(mg g^-1 fw) (mmol h^-1 g^-1 fw)

Root Leaves Root Shoot Root Shoot Root Shoot

Acaulospora morrowae 1.14 9.14 71.06 76.18 0.14 0.21 0.29 1.15

Gigaspora margarita 1.76 12.22 103.20 116.07 0.23 0.28 0.72 1.20

Glomus fasciculatum 1.58 11.79 94.24 99.33 0.20 0.25 0.54 1.16

G. macrocarpum 1.51 10.46 90.18 93.26 0.17 0.22 0.42 1.17

Sclerocystis rubiformis 1.42 10.17 82.16 87.20 0.15 0.19 0.34 1.18

Scutellospora calospora 1.92 14.02 110.15 125.56 0.27 0.35 0.78 1.24

Control 0.72 7.13 55.18 56.23 0.12 0.17 0.20 1.12 L.S.D. at P £ 0.05 0.25 2.23 0.12 0.14 0.02 0.02 _ _

Mathur and Vyas — Biochemical changes in Ziziphus xylopyrus by VA mycorrhizae

Table 2. Changes in PRO and PPO activities in roots of Z. xylopyrus as a result of infection by different VAM fungi.

Treatment PRO activity PPO activity Total-P Total phenol Catechin (Units mg^-1 protein) (DA₄₂₀/100 mg fw) (mg g^-1 dw) (% dw) (% dw)

Acaulospora morrowae 102.4 109.4 3.8 4.1 7.0

Gigaspora margarita 137.3 142.6 6.2 6.7 10.3

Glomus fasciculatum 120.5 130.2 5.4 6.0 9.3

G. macrocarpum 116.2 123.3 4.9 5.4 8.4

Sclerocystis rubiformis 112.4 118.5 4.3 4.9 7.9

Scutellospora calospora 178.0 183.6 7.6 7.9 11.2

Control 86.2 92.4 3.2 3.4 6.4

L.S.D. at P £ 0.05 19.5 14.2 1.2 1.2 0. 5

Jackson, M.L. 1973. Soil Chemical Analysis Prentice Hall of Indian Ltd., New Delhi, 574 pp.

Krishna, K.R. and D.J. Bagyaraj. 1984. Phenois in mycorrhizal roots of Arachis hypogea. Experientia 40L: 85_86.

Mahadevan, A. 1975. Methods in Physiological Plant Pathology. Sivakami Publication, Madras, India.

Mathur, N. and A. Vyas. 1995. Changes in isozme patterns of peroxidase and polyphenol oxidase by VAM fungi in roots of Ziziphus species. J. Plant Physiol. 145(4): 498_500.

McArthur, D.A.J. and N. R. Knowles. 1992. Resistance responses of potato to vesicular-arbuscular mycorrhizal fungi under varying abiotic phosphorus levels. Plant Physiol. 100: 341_351.

McArthur, D.A.J. and N. R. Knowles. 1993. Influence of vesicular-arbuscular mycorrhizal fungi on the response of potato to phosphorus deficiency. Plant Physiol. 101: 147_160.

Oliver, A.J., S.E. Smith, D.J.D. Nicholas, W. Wallace, and F.A. Smith. 1983. Activity of nitrate reductase in Trifolium subterraneum: Effects of mycorrhizal infection and phosphate nutrition. New Phytol. 94: 63_79.

Racusen, D. and M. Foote. 1965. Protein synthesis in dark grown bean levels. Can. J. Bot. 43: 817_824.

Schenck, N.C. and Y. Perez. 1987. Manual for the Identification of VA Mycorrhizal Fungi, pp. 11_14.

Shapiro, B.M. and E.R. Stadtman. 1970. Glutamine synthetase (Escherichia). Methods Enzymol. 17A: 910_922.

Smith, S.E., B.J. St. John, F.A. Smith, and D.J.D. Nicholas. 1985. Activity of glutamine synthetase and glutamate dehyrogenase in Allium cepa L. and Trifolium subterraneum L.: Effects of mycorrhizal infection and phosphate nutrition. New Phytol. 99: 211_227.

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pathogens (Mathur and Vyas, 1995). The considerable increase in PRO and PPO by S. calospora may be significant in protecting this fruit tree from attacking pathogens. Similarly, the increased catechin levels by the VAM inoculation (Table 2) may also enhance the pathogen defence mechanism of Z. xylopyrus. A nearly twofold increase in protein content was observed in leaves of Z. xylopyrus inoculated with S. calospora (Table 1). The higher protein accumulation in leaves of this plant may be advantageous when used as fodder for animals. Hence this VAM endophyte can also improve the nutritive value of Z. xylopyrus leaves.

It can be concluded that inoculation with S. calospora will cause elevation of assimilating enzymes most efficiently in Z. xylopyrus, which collectively will lead to increased biomass and more highly proteinaceous leafy fodder. It will also make the plant more resistant to pathogens as a result of increased PPO and PRO activities.

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Bray, H.G. and W.V. Thrope. 1954. Estimation of phenols. In D. Glick (ed.), Methods of Biochemical Analysis. vol. 1 Interscience Publishing Inc, New York, pp. 27_52.

Cliquet, J.B. and G.R. Stewart. 1993. Ammonia Assimilation in Zea mays L. infected with a vesicular arbuscular mycorrhizal fungus Glomus fasciculatum. Plant Physiol. 101: 865_871.

Gerdemann, J.W. and T.H. Nicolson. 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Br. Mycol. Soc. 46: 235_244.

Gulati, A., A. Gulati, S.D. Ravindernath, G. Satyanarayana, and D.N. Chakrabarty. 1993. Effect of blister blight of infusion quality in orthodoxtea. Indian Phytopathol. 46(2): 155_159.