Effects of Inoculation with Azospirillum Brasilense on Photosynthetic Enzyme Activities and Grain Yield in Maize

Effects of inoculation with Azospirillum brasilense on photosynthetic enzyme activities and grain yield in maize I Stancheva, I Dimitrov, N Kaloyanova, A Dimitrova, M Angelov To cite this version: I Stancheva, I Dimitrov, N Kaloyanova, A Dimitrova, M Angelov. Effects of inoculation with Azospir- illum brasilense on photosynthetic enzyme activities and grain yield in maize. Agronomie, EDP Sciences, 1992, 12 (4), pp.319-324. hal-00885478 HAL Id: hal-00885478 https://hal.archives-ouvertes.fr/hal-00885478 Submitted on 1 Jan 1992 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Plant-microorganism interactions Effects of inoculation with Azospirillum brasilense on photosynthetic enzyme activities and grain yield in maize I Stancheva1 I Dimitrov1 N Kaloyanova1 A Dimitrova1 M Angelov2 1N Poushkarov Institute of Soil Science and Agroecology, Shosses Bankja-7, 1080 Sofia; 2M Popov Institute of Plant Physiology, Bulgarian Academy of Science, Sofia, Bulgaria (Received 12 April 1991; accepted 16 January 1992) Summary — The influence of the inoculation of maize seeds with Azospirillum brasilense strain 1774 on accumulation of dry biomass, phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylases, nitrate-reductase activity and nitrogen fixing activity was best expressed at a nitrogen dose of 100 kg N ha-1. The inoculated plants showed a high intensity of dry biomass accumulation at the subsequent phenophases of maize development. The grain yield was similar in the noninoculated and inoculated plants with nitrogen fertilization treatment of 200 and 100 kg N ha-1. Zea mays (L) = maize Azospirillum brasilense / nitrogenase / phosphoenolpyruvate carboxylase / ribulose-1,5-bisphosphate carboxylase Résumé — Effets d’une inoculation de Azospirillum brasiliense sur l’activité des enzymes photo- synthétiques et le rendement en grain du maïs. L’influence de l’inoculation du maïs avec la souche 1774 d’Azospirillum brasilense sur l’accumulation de biomasse sèche, les activités phosphoénolpyruvate, ribulosediphosphate carboxylases, nitrate-réductase et l’activité fixatrice d’azote s’exprime mieux à une dose moyenne de fertilisation azotée du maïs. Les plantes inoculées ont montré une vitesse importante d’accumulation de biomasse sèche, dans les phases plus tardives du développement du maïs. Le rendement en grain était le même chez les plantes non-inoculées et inoculées respectivement à 200 et 100-1 kg ·ha d’a- zote. Zea mays (L) = maïs / Azospirillum brasilense / nitrogenase phosphoenolpyruvate carboxylase / ribulosediphosphate carboxylase INTRODUCTION from 10-30% (O’Hara et al, 1981; Pal and Ma- lik, 1981); nevertheless, the nitrogen fixation by associative bacteria in a temperate climate Inoculation of with brasi- plants Azospirillum seems to be negligible (Boddey et al, 1986). lense can result in a significant change in va- rious plant growth parameters, which may Okon (1985) evaluated the success of affect crop yield. Visible changes have been Azospirillum inoculation and concluded that observed in total plant dry weight, in the positive effects on yield were obtained in ap- amount of nitrogen in shoots and grains and proximately 65% of all field experiments. How- in the grain weight (O’Hara et al, 1981; Al- ever, negative results or no effect of brecht et al, 1983; Bashan and Levanony, inoculation on grain yield have been reported 1990). (Albrecht et al, 1983; Smith et al, 1984; Harris et al, 1989). The effects of inoculation with Azospirillum bacteria on total yield increase in field grown The highest yield increases were obtained plants have been found to generally range when the levels of nitrogen fertilization were suboptimal for normal plant development (Bal- (mM): HEPES-KOH (pH=8.0), 20; MgCl2, 20; dani and Döbereiner, 1981; O’Hara et al, DTT, 10; NaHCO3, 20; NaH14CO3, 1003 mm (spe- 1987). The normal development of maize after cific activity 1.67 MBq μmol-1) and 0.43 cm homo- The RuBPC was activated at 25°C for 10 inoculation was obtained at a nitrogen level genate. min and the reaction was with which was 10-20% of the level enzyme stopped optimum (Ste- 3 N HCl. phen et al, 1982). PEPC activity was determined in the reaction The aim of our was to deter- investigation mixture (1 cm3) containing (mM): HEPE-KOH, 20; mine the influence of the inoculation of maize MgCl2, 10; DTT, 10; NaDH (disodium salt), 0.4; seeds with Azospirillum brasilense on photo- NaHCO3, 20; NaH14CO3, 1003 mm and 0.43 cm ho- synthetic activity and grain yield at different mogenate. doses. nitrogen The PEPC was activated at 30°C for 5 min and the enzyme reaction started by addition of PEP (Merck). The radiometric activity of carboxylases was determined in a scintillator counter. Materials and Methods liquid Nitrogenase activity was determined by the C2H-reduction method on the excised roots (Dö- The roots were under and conditions bereiner, 1978). placed po- Organisms growth lyethylene bags and exposed to acetylene- enriched air (10% acetylene) for 24 h. The gas were into a The investigations were made on the basis of 2 samples injected gas chromatograph fitted with a flame ionisation detector. The years of field experiments including 3 levels of ni- gas were and the of trogen fertilization (0, 100, 200 kg N-1) ha 55 000 peaks analysed amount ethylene was calibrated a commercially plants per ha and a nondeficient water regime. The produced against prepared ethylene standard of known ethylene maize plants, Zea mays (L) hybrid H 708, were concentration. grown on leached cinnamonic forest soil whose or- ganic matter content was 1.7-1.0% and pH 6.6. Nitrate-reductase activity (E.C.1.6.6.1) was measured to et al Ten A randomized block design with 4 replicates was according Klepper (1971). were taken for matter deter- used to test inoculation with Azospirillum. Each plants per variant dry mination. The data were to plot was 64 m2 in area and consisted of 12 rows. experimental subjected standard statistical The seeds were inoculated with Azospirillum bra- procedure. silenze strain 1774 (UQM, 13 collection) donated by Dr LI Sly from the University of Queensland, Australia. Azospirillum brasilense bacteria were grown on semi-liquid malate medium (Albrecht and Results and Discussion Okon, 1980). Inoculation was performed before sowing with inoculum containing 108 colony-forming units ml-1 and 400 ml per each kg of seeds. The data of biomass accumulation during the Microbial tests were performed in root samples different phenophases are shown in figure 1. taken from 3 plants chosen at random from each An even accumulation of dry biomass was ob- plot. Isolation, identification and counting of Azos- served till the milk ripeness phase in non-fer- were to Okon et al pirillum performed according tilized variants in the non-inoculated as well (1977). as in the inoculated plants. In the non-inoculated fertilized variants, plants acquired a higher forming rate of dry biomass between the Enzyme assay tasseling and milk ripeness phases. In contrast to non-inoculated plants, inoculated Ribulose-1,5-bisphosphate carboxylase RuBPC plants showed higher forming rate of dry bio- (E.C.4.1.1.31) activity was measured according to mass after the milk ripeness phase. This trend the method of Angelov (Angelov et al, 1987). The was observed at the 3 levels of nitrogen fer- leaf tissue (0.5 g fresh matter) was homogenized tilization, but was most significant at at 4 °C with 5 cm3 buffer containing (mM) : HEPES- 100 kg N ha-1. The growth in dry biomass of KOH (pH = 8.0), 50; sorbitol, 330; KNO3, 2; EDTA, the maize inoculated with Azospirillum at the 2; MgCl2,1; K2HPO4, 0.5; NaCl, 20; isoascorbate subsequent phenophases has been reported (sodium salt), 2; Polyclar AT (10% m/v) was added by other authors (Pidello, 1981; Okon, 1982; to each sample. The was passed homogenate Barber et al, 1986). through 4 layers of cheesecloth and the filtrate was centrifuged at 10 000 g for 10 min at 0°C. The The dry biomass increased with nitrogen reaction mixture (1 cm3) for RuBPC contained doses independently of the inoculation (fig 1). On the other hand, the influence of inoculation In the inoculated variants with 100 kg N ha-1 on dry biomass accumulation was best ex- maximum leaf area index changed from 3.3 pressed at 100 kg N ha-1, since significant dif- to 4.4, Pn from 6.26 to 7.17 and the plant ferences were observed in total plant dry height from 2.74 to 2.75. The values of these weight (fig 1). There was no effect of inocula- parameters in the 2 variants considered do tion on accumulation of dry biomass in va- not differ significantly. riants without nitrogen. The investigation of Nitrogen fixing activity was detected in all the influence of inoculation on yield produc- variants at grain filling phase (table II). Accor- tion has very important practical implications. ding to some authors (Xavier et al, 1982; Rao The statistically reliable differences in yield and Venkateswarlu, 1985), root extracts from production are shown under the influence of plants collected at this growth stage stimula- the fertilizing factor only (table I). The in- ted Azospirillum growth to a great extent. The fluence of inoculation and the interaction be- high intensity of dry biomass accumulation in tween both factors on yield production has not inoculated plants after the milk ripeness been proven mathematically.

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