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Effects of Inoculation of Maize and Species of Tribe Triticeae with Azospirillum brasilense
J. Plant Pbysiol.
Vol. 140. pp. 550-552 {1992}
Effects of Inoculation of Maize and Species of Tribe Triticeae with Azospirillum brasilense IRA STANCHEVA and NICKOLAS DINEV N. Poushkarov Institute of Soil Science and Agroecology, P.O. Box 1369, 7 Shosse Bankja str., Sofia 1080, Bulgaria Received November 28,1991 . Accepted March 12, 1992
Summary
The interaction between Azospirillum bacteria and the maize root system increased the plant biomass and total nitrogen content. Association between species of the tribe Triticeae and Azospirillum brasilense differed according to the genome varieties of the wheats. Interaction between A. brasilense and wheat plants was most efficient in Triticum aestivum and Dazypyrum villosum. An increase in biomass, total nitrogen and carotenoid content was observed in those species after inoculation.
Key words: Triticeae, Zea mays, Azospirillum brasilense, pigments.
Introduction
Investigations on the effects of inoculation of cereals with nitrogen-fixing bacteria have been very intensive for the last 10 years. The specific relationship between nitrogen-fixing bacteria and crops depends on their genetic features (Stephen et al., 1982), which has been observed among different wheat varieties (Millet et al., 1985). Specific genetic properties considerably influence the degree of effectivity of associated fixation (Rodinjuk, 1988). Information about host-plant and bacteria interactions is very important because of the insufficient results in this area. The aim of our investigation was to establish some physiological changes in wheat and maize plants after inoculation with Azospirillum brasilense and to define the influence of genotypic properties on plant-bacteria interaction.
losum (L.) Candargi (2n - 14, V-genome) and Triticum aestivum L. (2n = 42, ABD-genome) cv. Chinese Spring were studied. The maize plants (one plant per pot) were grown in 5-L plastic pots until the 8-9 leaf phase, and wheat (5 seedlings per pot) in l-L plastic pots until the tiller phase. The plants were grown on complete Arnon-Hoagland solution at a nitrogen concentration of 126,5 mg per kg sand, where NO)" in KN0 3 and Ca(N03h and NHt in NH4H 2P0 4 and (NH4)~o . 4H 20 were included. The experiments were conducted in a controlled environmental chamber with a day temperature of 25 oC, night temperature of 18°C, photoperiod of 16 hours, relative humidity of 75 % and visible radiation of 335 W . m -2 and 150 W . m -2 for maize and wheat, respectively. The plants were irrigated daily to maintain a moisture content of 70 %. The seeds were inoculated with Azospirillum brasilense T arrand et al., strain 1774. Azospirillum brasilense bacteria were grown on a semi-liquid malate medium (Albrecht and Okon, 1980). Before sowing, seeds in the pots were inoculated with 5 mL of a suspension of A. brasilense (10 9 colony-forming units per mL). After extraction with 80 % acetone the content of plastid pigments was determined using Vernon's formula and the total nitrogen content was established after Kjeldahl.
Material and Methods Three repetitive experiments were carried out for maize and five for plants of tribe Triticeae. One species of maize (Zea mays L., Hybrid H-708) and six species of tribe Triticeae, i.e. Triticum boeoticum Boiss. (2n = 14, A-genome), Aegilops speltoides Tausch. (2n ~ 14, Bgenome), Triticum dicoccum Schuebl. (2n = 28, AB-genome), Tri· ticum timopheevii Zhuk. (2n = 28, AG-genome), Dazypyrum vil© 1992 by Gustav Fischer Verlag, Stuttgart
Results and Discussion
Plastid pigment content was determined in leaves (Table 1). Significantly higher values of chlorophyll «a» «b» and of carotenoids were found in inoculated plants of T. aestivum,
Inoculation with Azospirillum Table 1: Chlorophyll and carotenoid content in leaves of inoculated and non-inoculated plants of Zea mays L. and tribe Triticeae (g kg- I F.W .). Species
Non-inoculated plants Chlorophyll «a» «b»
Zea mays L. H-708 LSD-P=5%
Carotenoids
Chlorophyll «a» «b»
Carotenoids
1.131
0.480
1.744 1.120 0.471 0.095 0.082 0.056
Table 2: Influence of inoculation with Azospirillum brasilence on accumulation of fresh biomass in plants of Zea mays L. and tribe Triticeae(g F.W.• plant-I).
Zea mays L. H-708 LSD-P=5%
sf
Inoculated plants
Tribe Triticeae 1.145 0.747 0.355 1.559 0.972 0.472 T. boeoticum 0.531 0.354 0.156 Ae. speltoides 1.106 0.745 0.377 1.730 1.115 0.616 T. dicoccum 1.570 1.015 0.566 1.332 0.845 0.424 T. timopheevii 1.192 0.734 0.409 0.674 0.443 0.516 0.903 0.607 0.304 D. villosum 0.955 0.680 0.316 T. aestivum 0.803 0.566 0.253 0.062 0.066 0.048 LSD*-P=5% * Differences of values within the species are not significant. Presented LSD expresses differences among the species of tribe Triticeae.
Species
:f
6~
:<1 :lrrrn]. , , :. 2
,
:b ,'
.'
.'
A-genoll'le
1.683
Shoots InocuNon-inlated oculated 138.33
30.19
190.00
551
Roots InocuNon-inoculated lated 46.00
7.80
55.00
Tribe Triticeae 0.244 0.389 0.166 T. boeoticum 0.486 0.030 Ae. speltoides 0.065 0.347 0.286 2.147 0.575 0.541 T. dicoccum 1.825 0.417 0.574 1.363 T. timopheevii 1.425 0.077 0.168 0.519 D. villosum 0.286 0.544 0.830 2.649 2.069 T. aestivum 0.081 LSD*-P=5% 0.071 * Differences of values within the species are not significant. Presented LSD expressed differences among the speices of tribe Triticeae.
T. dicoccum and T. boeoticum than in non-inoculated controls. In plants of Ae. speltoides the plastid pigment content was lower in inoculated variants. There were no differences in plastid pigment contents of maize leaves in regard to inoculation. In D. villosum plants chlorophyll values were lower while carotenoid values were higher than in controls. In T. timopheevi~ only chlorophyll «b» increased. Opinions differ regarding the relationship between inoculation with nitrogen-fixing bacteria and chlorophyll content. According to Nur et al. (1981) there is a direct relationship between nitrogen-fixing activity and carotenoid content. It has been proposed that carotenoids playa role in the protection of Azospirillum nitrogenase against oxygen radicals. Inoculation with nitrogen-fixing bacteria influences biomass accumulation (Table 2). This influence was greatest in
'-o-"-"--~"":;;-'~~':;-'':''cc:~';;-''-::: ....'-.x==---"'~~~
Fig. 1: Total nitrogen content in inoculated and non-inoculated plants of Zea mays L. and tribe Triticeae. a: Non-inoculated, b: Inoculated.
D. villosum with a two-fold increase in shoot and root biomass in inoculated variants compared with non-inoculated ones. In the other variants, Ae. speltoides, T. dicoccum and T. aestivum, significant increases in biomass by inoculation were shown. Only in T. boeoticum shoot biomass was lower but root biomass was higher in inoculated plants. Our results show that an increase in biomass was observed in all of the species containing the B-genome (Ae. speltoides, T. dicoccum, T. aestivum). One reason for this effect may be an improved energetic status because of the enhanced photosynthetic activity, described in other reports (Morgenstern and Okon, 1987). There are contradictory reports about the influence of inoculation on root biomass and development. Some authors (Zimmer and Bothe, 1988; Alexander and Zuberer, 1988) suggested that nitrogen-fixing bacteria have a beneficial effect on root length, number of root hairs and root weight, which may be due to excretion of phytohormones after inoculation. Other authors reported that inoculation influenced root length and number of root hairs but not root weight (Tien et al., 1979). Our results show an increase in root biomass by Azospirillum in all experimental variants except for
T. dicoccum.
The maize plants inoculated with Azospirillum brasilense showed high total nitrogen content in shoots. A similar effect was observed in some wheat species - T. aestivum, Ae. speltoides and D. villosum. There were no significant differences in nitrogen content between inoculated and non-inoculated plants of T. dicoccum (Fig. 1). An increase of nitrogen content in shoots reflected favourable nutrient conditions in inoculated variants. Enhanced proliferation of the root system could promote increased mineral uptake and consequently increased dry biomass accumulation (Lin et aI., 1983; Okon and Kapulnik, 1986). An association between Azospirillum bacteria and the maize root system at a nitrogen dose lower than the optimum led to an increase in plant biomass and total nitrogen content. The relationship between A. brasilense and wheat plants was most efficient in plants of T. aestivum and D. villosum. In both species increased biomass, total nitrogen and carotenoid content were seen following inoculation. The effect of inoculation on other variants of wheat was less pronounced. Acknowledgements
The authors wish to thank Mrs. L. Nikolova for technical help and statistical analysis.
552
IRA STANCHEVA and NICKOLAS DINEV
This work was supported by a grant from the Bulgarian Agricultural Academy.
References ALBRECHT, S. L. and Y. OKON: Cultures of Azospirillum. Methods of Enzymology 69,740-749 (1980). ALEXANDER, D. L. and D. A. ZUBERER: Impact of Soil Environmental Factors on Rates of Nrfixation Associated with Roots of Intact Maize and Sorghum Plants. Plant and Soil 111, 303 - 309 (1988). LIN, W., Y. OKON, and R. W. HARDY: Enhanced Mineral Uptake by Zea Mays and Sorghum Bicolor Roots Inoculated with Azospiril· lum brasilense. Applied and Environmental Microbiology 45, 1775-1779 (1983). MiLLET, E., Y. AVIVI, and M. FELDMAN: Effects of Rhizospheric Bacteria on Wheat Yield under Field Condition. Plant and Soil 86, 347 -355 (1985). MORGENSTERN, E. and Y. OKON: Promotion of Plant Growth and NO} and Rb+ Uptake in Sorghum bieolor x Sorghum sudanense
Inoculated with Azospirillum brasilense. Cd. Arid Soil Research and Rehabilitation 1, 211-217 (1987). NUR, 1., Y. L. STEINITZ, Y. OKON, and Y. HENIS: Carotenoids Composition and Function in Nitrogen-fixing Bacteria of Genus azospirillum. Journal of Genetics Microbiology 122, 27 -32 (1981). OKON, Y. and Y. KApULNIK: Development and Function of Azospirillum-inoculated Roots. Plant and Soil 90, 3-16 (1986). RODINJUK, 1. S.: Influence of Mineral Nitrogen on Rhizosphere Nitrogen Fixation by Agricultural Plants. Vsesojuznie Tezisi Dokladov, Pushtchino, pp. 114-115 (1988) (Russian). STEPHEN, W. E., M. A. ANDERSON, and W. J. BRILL: Screening and Selection of Maize to Enhance Associative Bacterial Nitrogen Fixation. Plant Physiol. 2, 236-241 (1982). TIEN, T. M., M. H. GASKINS, and D. H. HUBBELL: Plant Growth Substances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum amerieanum L.). Applied and Environmental Microbiology 37, 1016-1024 (1979). ZIMMER, W. and H. BOTHE: The Phytohormonal Interactions between Azospirillum and Wheat. Plant and Soil 110, 239-247 (1988).
Vol. 140. pp. 550-552 {1992}
Effects of Inoculation of Maize and Species of Tribe Triticeae with Azospirillum brasilense IRA STANCHEVA and NICKOLAS DINEV N. Poushkarov Institute of Soil Science and Agroecology, P.O. Box 1369, 7 Shosse Bankja str., Sofia 1080, Bulgaria Received November 28,1991 . Accepted March 12, 1992
Summary
The interaction between Azospirillum bacteria and the maize root system increased the plant biomass and total nitrogen content. Association between species of the tribe Triticeae and Azospirillum brasilense differed according to the genome varieties of the wheats. Interaction between A. brasilense and wheat plants was most efficient in Triticum aestivum and Dazypyrum villosum. An increase in biomass, total nitrogen and carotenoid content was observed in those species after inoculation.
Key words: Triticeae, Zea mays, Azospirillum brasilense, pigments.
Introduction
Investigations on the effects of inoculation of cereals with nitrogen-fixing bacteria have been very intensive for the last 10 years. The specific relationship between nitrogen-fixing bacteria and crops depends on their genetic features (Stephen et al., 1982), which has been observed among different wheat varieties (Millet et al., 1985). Specific genetic properties considerably influence the degree of effectivity of associated fixation (Rodinjuk, 1988). Information about host-plant and bacteria interactions is very important because of the insufficient results in this area. The aim of our investigation was to establish some physiological changes in wheat and maize plants after inoculation with Azospirillum brasilense and to define the influence of genotypic properties on plant-bacteria interaction.
losum (L.) Candargi (2n - 14, V-genome) and Triticum aestivum L. (2n = 42, ABD-genome) cv. Chinese Spring were studied. The maize plants (one plant per pot) were grown in 5-L plastic pots until the 8-9 leaf phase, and wheat (5 seedlings per pot) in l-L plastic pots until the tiller phase. The plants were grown on complete Arnon-Hoagland solution at a nitrogen concentration of 126,5 mg per kg sand, where NO)" in KN0 3 and Ca(N03h and NHt in NH4H 2P0 4 and (NH4)~o . 4H 20 were included. The experiments were conducted in a controlled environmental chamber with a day temperature of 25 oC, night temperature of 18°C, photoperiod of 16 hours, relative humidity of 75 % and visible radiation of 335 W . m -2 and 150 W . m -2 for maize and wheat, respectively. The plants were irrigated daily to maintain a moisture content of 70 %. The seeds were inoculated with Azospirillum brasilense T arrand et al., strain 1774. Azospirillum brasilense bacteria were grown on a semi-liquid malate medium (Albrecht and Okon, 1980). Before sowing, seeds in the pots were inoculated with 5 mL of a suspension of A. brasilense (10 9 colony-forming units per mL). After extraction with 80 % acetone the content of plastid pigments was determined using Vernon's formula and the total nitrogen content was established after Kjeldahl.
Material and Methods Three repetitive experiments were carried out for maize and five for plants of tribe Triticeae. One species of maize (Zea mays L., Hybrid H-708) and six species of tribe Triticeae, i.e. Triticum boeoticum Boiss. (2n = 14, A-genome), Aegilops speltoides Tausch. (2n ~ 14, Bgenome), Triticum dicoccum Schuebl. (2n = 28, AB-genome), Tri· ticum timopheevii Zhuk. (2n = 28, AG-genome), Dazypyrum vil© 1992 by Gustav Fischer Verlag, Stuttgart
Results and Discussion
Plastid pigment content was determined in leaves (Table 1). Significantly higher values of chlorophyll «a» «b» and of carotenoids were found in inoculated plants of T. aestivum,
Inoculation with Azospirillum Table 1: Chlorophyll and carotenoid content in leaves of inoculated and non-inoculated plants of Zea mays L. and tribe Triticeae (g kg- I F.W .). Species
Non-inoculated plants Chlorophyll «a» «b»
Zea mays L. H-708 LSD-P=5%
Carotenoids
Chlorophyll «a» «b»
Carotenoids
1.131
0.480
1.744 1.120 0.471 0.095 0.082 0.056
Table 2: Influence of inoculation with Azospirillum brasilence on accumulation of fresh biomass in plants of Zea mays L. and tribe Triticeae(g F.W.• plant-I).
Zea mays L. H-708 LSD-P=5%
sf
Inoculated plants
Tribe Triticeae 1.145 0.747 0.355 1.559 0.972 0.472 T. boeoticum 0.531 0.354 0.156 Ae. speltoides 1.106 0.745 0.377 1.730 1.115 0.616 T. dicoccum 1.570 1.015 0.566 1.332 0.845 0.424 T. timopheevii 1.192 0.734 0.409 0.674 0.443 0.516 0.903 0.607 0.304 D. villosum 0.955 0.680 0.316 T. aestivum 0.803 0.566 0.253 0.062 0.066 0.048 LSD*-P=5% * Differences of values within the species are not significant. Presented LSD expresses differences among the species of tribe Triticeae.
Species
:f
6~
:<1 :lrrrn]. , , :. 2
,
:b ,'
.'
.'
A-genoll'le
1.683
Shoots InocuNon-inlated oculated 138.33
30.19
190.00
551
Roots InocuNon-inoculated lated 46.00
7.80
55.00
Tribe Triticeae 0.244 0.389 0.166 T. boeoticum 0.486 0.030 Ae. speltoides 0.065 0.347 0.286 2.147 0.575 0.541 T. dicoccum 1.825 0.417 0.574 1.363 T. timopheevii 1.425 0.077 0.168 0.519 D. villosum 0.286 0.544 0.830 2.649 2.069 T. aestivum 0.081 LSD*-P=5% 0.071 * Differences of values within the species are not significant. Presented LSD expressed differences among the speices of tribe Triticeae.
T. dicoccum and T. boeoticum than in non-inoculated controls. In plants of Ae. speltoides the plastid pigment content was lower in inoculated variants. There were no differences in plastid pigment contents of maize leaves in regard to inoculation. In D. villosum plants chlorophyll values were lower while carotenoid values were higher than in controls. In T. timopheevi~ only chlorophyll «b» increased. Opinions differ regarding the relationship between inoculation with nitrogen-fixing bacteria and chlorophyll content. According to Nur et al. (1981) there is a direct relationship between nitrogen-fixing activity and carotenoid content. It has been proposed that carotenoids playa role in the protection of Azospirillum nitrogenase against oxygen radicals. Inoculation with nitrogen-fixing bacteria influences biomass accumulation (Table 2). This influence was greatest in
'-o-"-"--~"":;;-'~~':;-'':''cc:~';;-''-::: ....'-.x==---"'~~~
Fig. 1: Total nitrogen content in inoculated and non-inoculated plants of Zea mays L. and tribe Triticeae. a: Non-inoculated, b: Inoculated.
D. villosum with a two-fold increase in shoot and root biomass in inoculated variants compared with non-inoculated ones. In the other variants, Ae. speltoides, T. dicoccum and T. aestivum, significant increases in biomass by inoculation were shown. Only in T. boeoticum shoot biomass was lower but root biomass was higher in inoculated plants. Our results show that an increase in biomass was observed in all of the species containing the B-genome (Ae. speltoides, T. dicoccum, T. aestivum). One reason for this effect may be an improved energetic status because of the enhanced photosynthetic activity, described in other reports (Morgenstern and Okon, 1987). There are contradictory reports about the influence of inoculation on root biomass and development. Some authors (Zimmer and Bothe, 1988; Alexander and Zuberer, 1988) suggested that nitrogen-fixing bacteria have a beneficial effect on root length, number of root hairs and root weight, which may be due to excretion of phytohormones after inoculation. Other authors reported that inoculation influenced root length and number of root hairs but not root weight (Tien et al., 1979). Our results show an increase in root biomass by Azospirillum in all experimental variants except for
T. dicoccum.
The maize plants inoculated with Azospirillum brasilense showed high total nitrogen content in shoots. A similar effect was observed in some wheat species - T. aestivum, Ae. speltoides and D. villosum. There were no significant differences in nitrogen content between inoculated and non-inoculated plants of T. dicoccum (Fig. 1). An increase of nitrogen content in shoots reflected favourable nutrient conditions in inoculated variants. Enhanced proliferation of the root system could promote increased mineral uptake and consequently increased dry biomass accumulation (Lin et aI., 1983; Okon and Kapulnik, 1986). An association between Azospirillum bacteria and the maize root system at a nitrogen dose lower than the optimum led to an increase in plant biomass and total nitrogen content. The relationship between A. brasilense and wheat plants was most efficient in plants of T. aestivum and D. villosum. In both species increased biomass, total nitrogen and carotenoid content were seen following inoculation. The effect of inoculation on other variants of wheat was less pronounced. Acknowledgements
The authors wish to thank Mrs. L. Nikolova for technical help and statistical analysis.
552
IRA STANCHEVA and NICKOLAS DINEV
This work was supported by a grant from the Bulgarian Agricultural Academy.
References ALBRECHT, S. L. and Y. OKON: Cultures of Azospirillum. Methods of Enzymology 69,740-749 (1980). ALEXANDER, D. L. and D. A. ZUBERER: Impact of Soil Environmental Factors on Rates of Nrfixation Associated with Roots of Intact Maize and Sorghum Plants. Plant and Soil 111, 303 - 309 (1988). LIN, W., Y. OKON, and R. W. HARDY: Enhanced Mineral Uptake by Zea Mays and Sorghum Bicolor Roots Inoculated with Azospiril· lum brasilense. Applied and Environmental Microbiology 45, 1775-1779 (1983). MiLLET, E., Y. AVIVI, and M. FELDMAN: Effects of Rhizospheric Bacteria on Wheat Yield under Field Condition. Plant and Soil 86, 347 -355 (1985). MORGENSTERN, E. and Y. OKON: Promotion of Plant Growth and NO} and Rb+ Uptake in Sorghum bieolor x Sorghum sudanense
Inoculated with Azospirillum brasilense. Cd. Arid Soil Research and Rehabilitation 1, 211-217 (1987). NUR, 1., Y. L. STEINITZ, Y. OKON, and Y. HENIS: Carotenoids Composition and Function in Nitrogen-fixing Bacteria of Genus azospirillum. Journal of Genetics Microbiology 122, 27 -32 (1981). OKON, Y. and Y. KApULNIK: Development and Function of Azospirillum-inoculated Roots. Plant and Soil 90, 3-16 (1986). RODINJUK, 1. S.: Influence of Mineral Nitrogen on Rhizosphere Nitrogen Fixation by Agricultural Plants. Vsesojuznie Tezisi Dokladov, Pushtchino, pp. 114-115 (1988) (Russian). STEPHEN, W. E., M. A. ANDERSON, and W. J. BRILL: Screening and Selection of Maize to Enhance Associative Bacterial Nitrogen Fixation. Plant Physiol. 2, 236-241 (1982). TIEN, T. M., M. H. GASKINS, and D. H. HUBBELL: Plant Growth Substances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum amerieanum L.). Applied and Environmental Microbiology 37, 1016-1024 (1979). ZIMMER, W. and H. BOTHE: The Phytohormonal Interactions between Azospirillum and Wheat. Plant and Soil 110, 239-247 (1988).