Azide resistant mutants of Acetobacterdiazotrophicus and Azospirillum brasilense increase yield and nitrogen content of cotton

Abstract

Evolution of symbiotic plant-microbe interactions has provided mankind a powerful and environment-friendly means to increase yield of agricultural crops. Here, we report that some azide resistant mutants of two microbial strains can significantly enhance the productivity of cotton varieties, as an attractive and cheap biological substitute of chemical fertilizers, for improved yield of an important cash crop, without any untoward impacts. Sodium azide resistant mutants were isolated from each strain of Azospirillum brasilense and Acetobacter diazotrophicus on different concentrations of sodium azide ranging from 5–60µg/ml. These azide resistant mutants were assessed for their performance on cotton (varieties H-117, HD-123) for various parameters. Inoculation of cottonseeds with mutants obtained better results than inoculation with their respective parental strains. Azide resistant mutants, when used as biofertilizers, showed increased plant height, early flowering, more yield, and high biomass and total nitrogen content. They also increased, in cotton genotypes, the indole acetic acid production and ammonia excretion due to high nitrogenase activity.

Keywords:

• Acetobacter diazotrophicus
• Azospirillum brasilense
• sodium azide resistant mutants
• cotton

Introduction

With the advent of green revolution technologies, modern agriculture is getting more and more dependent upon the steady supply of synthetic inputs (mainly fertilizers), which are products of fossil fuels such as coal and petroleum. Adverse effects are being noticed due to the excessive and imbalanced use of these synthetic inputs. This situation has necessitated identifying harmless and natural product inputs like biofertilizers. Use of biofertilizers in crop cultivation will most certainly help in safeguarding the soil health and also the quality of crop products. Efforts at extending nitrogen(N₂)-fixing ability to important non-leguminous crops such as cereals and cash crops have long been a major goal of workers in the field of biological nitrogen fixation.

Resistance to azide can be used as a potentially useful method to select efficient strains. Sodium azide has been used as a chemical mutagen by various researchers (Gordon & Brill 1972, Yadav 1999) for strain selection. Azide resistant mutants of Azotobacter chroococcum have been found with improved N₂-fixing ability in cotton (Sharma et al. 2002). The exact mechanism by which azide resistant strains fix more nitrogen, however, is not clear. Sodium azide is a potential inhibitor of cytochrome oxidase and also acts as an alternate substrate for the enzyme nitrogenase.

One approach for achieving this goal has involved the isolation and characterization of azide resistant mutant of N₂ -fixing bacteria such as Acetobacter diazotrophicus and Azospirillum brasilense and assessing their performance on two cotton genotypes (HD-123 and H-117).

Materials and methods

A strain (35–47) of A. diazotrophicus and one of A. brasilense (FS), procured from the Department of Microbiology, CCSHAU, Hisar, India, were used for the present study. Seeds of two cotton cultivars, namely Gossypium hirsutum cv H117 and G. herbaceum cv HD-123, were obtained from the Cotton Section, Department of Plant Breeding, CCSHAU, Hisar. Semi solid N-free medium (LGI medium, Cavalcante & Doberenier 1988) was used for isolation of Acetobacter and for testing resistance to sodium azide, while malate medium (Sadasivan & Neyra 1995) was used for culturing and isolation of mutants of A. brasilense. Isolated azide resistant mutants were further investigated by spotting on their respective culture media containing a particular concentration of azide. The nitrogenase activity (acetylene reduction activity, ARA) of strains and mutants was assayed by growing the parent microbial strain and mutant on Burk's nitrogen-free medium slants (Quispel 1974). Indole acetic acid (IAA) production by parent strains and mutants was assayed by the method as described by Hartmann et al. (1983) using appropriate broths supplemented with 100 µg/ml tryptophan. IAA was determined in culture supernatant by Salkowski's method as reported by Glickmann and Dessaux (1995). Ammonia excretion by A. diazotrophicus and A. brasilense strains and mutants was studied by growing cultures in LGI broth and malate broth, respectively, and ammonia released was determined in the supernatant by using the method of Chaney and Marbach (1962). Nitrogen content of roots, stems, leaves and carpels was separately estimated by the micro-Kjeldhal's method (Association of Official Agricultural Chemists [AOAC] 1970). Cottonseed yield per plant was calculated to assess the overall performance of mutants as compared to parental strains.

Results and discussion

Azide resistant microbial mutants

The parent strains of A. diazotrophicus (35–47) and A. brasilense (FS) were tested to determine the minimal inhibitory concentration (MIC) of sodium azide by growing cells in the appropriate liquid media containing different concentrations of sodium azide. Mutants were found to have higher value of MIC as compared to parent strain. Similar results in Azotobacter have been reported by Sharma et al. (2002) and in Rhizobium by Yadav and Vashishat (1986). Three mutants of A. brasilense (FS D-B, FS D-m, and FS D-s) and one mutant of Acetobacter diazotrophicus (35–47 D) were named on the basis of their colony size (Table I).

Table I Azide resistance, acetylene reductase activity (ARA), indole acetic acid production (IAA) and ammonia excretion (AE) in parents and mutant strains of Acetobacter diazotrophicus and Azospirillum brasilense.

Strains/mutants	Conc. of sodium azide (µg/ml)	ARA (n moles c2h2h^−1tube^−1)	IAA (µg/ml)	Ammonia excretion (µg/ml)
35–47	5	1.11	68.31	60.76
35–47 D	15	6.21	76.31	66.19
FS	15	1.85	16.70	32.41
FS D-B	60	8.01	43.19	46.66
FS D-m	60	6.58	32.01	44.49
FS D-s	60	4.44	19.34	35.81
CD	–	0.03	–	–

Nitrogenase activity

Nitrogenase activity of azide resistant mutants was higher when compared with ARA activity of their respective parent strains. Similarly, Sharma et al. (1997) observed that ARA of the majority of azide resistant mutants of R. loti was higher than the parental strain NZP2037. Thus, a correlation exists between resistance to azide and Nitrogenase activity. When we compared the relative performance of the two bacteria, Azospirillum showed a better performance than Acetobacter, both in parental strains and mutants demonstrating better competitive ability in the former than the later (Table I). It can be safely surmised that the better these beneficial bacteria compete in the soil microbiota, more will their hormone production and more will be their colonies survive to effect improved nitrogen fixation for the host plant, and hence enhanced cotton crop productivity. A similar general conclusion can be drawn for other plant-microbe symbionts as well.

Indole acetic acid (IAA) production

IAA production was estimated up to the 27th day. IAA production was higher in mutants as compared to their respective parent strains. The pattern of IAA production was found to be at maximum on the 6th day of growth and then it exhibited a decreasing trend up to the 15th day. Beyond this day values became constant for both strains and mutants (Table I). A similar pattern was also found in Azotobacter by Sharma et al. (2002) and Brown and Walker (1970). Acetobacter diazotrophicus is an IAA-producing bacterium, isolated from sugarcane cultivars of Mexico; it has been suggested that biosynthesis of IAA by bacteria could promote rooting and improve sugarcane growth by direct effect on metabolic processes, in addition to their role in N₂ fixation (Stattar & Gaur 1987, Barbieri & Galli 1993, Fuentes-Ramirez et al. 1993, Leinhos & Vacek 1994). Phytohormone synthesized by Azospirillum influenced the root hair development, respiration rate, metabolism and root proliferation, which in turn resulted in better mineral uptake in the inoculated plant (Bar & Okon 1993).

Thus, it is conceivable that the endophytic and associative bacteria may improve plant productivity both by the involvement of plant growth regulating substances and by supplying nitrogen. In the present study, we observed that IAA production in Azospirillum lagged behind that of Acetobacter by as yet unknown mechanism.

Ammonia excretion

Ammonia excretion was higher in mutants as compared to their respective parent strains. Ammonia excretion was at a maximum on the 6th day and decreased after the 21st day of incubation (Table I). Narula et al. (1999) demonstrated that EDA mutant E-12 excreted higher amounts of ammonia in nitrogen-free media in comparison to parent A. chroococcum MAC 27 on the 8th day, but decreased afterwards. Similarly, ammonia excretion was very high in mutants of A. chroococcum MSx9D-B, MSx9D-m and HT-54 (2) D-s on the 15th day (Sharma et al. 2002). Ammonia excretion was higher in Acetobacter than Azospirillum.

Thus, ammonia excretion seems to be the result of nitrogenase activity as suggested by Raj Kumar and Lakshmanan (1995). Decrease in ammonia excretion with further incubation might be due to repression of nitrogenase, which further stops reduction of nitrogen. Repression of nitrogenase by higher concentration of combined nitrogen has also been reported in Azospirillum by Ahmed (1978). Similarly Burris et al. (1991) reported that in the presence of ammonia A. diazotrophicus showed a slow growth.

Nitrogen content

The percentage of nitrogen and the total nitrogen content of roots, stems, carpels and leaves of each plant treated with parent strains and mutants were estimated in both cotton varieties. There was a significantly higher value of nitrogen content in the plants treated with mutants as compared to the plants treated with their respective parent strains in both varieties. Nitrogen content was higher in plants inoculated with Acetobacter than in plants inoculated with Azospirillum (Table II).

Table II Effect of parental strain/mutants of Acetobacter diazotrophicus and Azospirillum brasilense on nitrogen content of carpel, leaves, stem and root in cotton genotypes.

Strain/mutants	Total nitrogen content
	Root		Leaves		Stem		Carpel
	HD-123	H-117	HD-123	H-117	HD-123	H-117	HD-123	H-117
35–47	3.12	3.64	8.79	11.62	8.98	11.13	5.59	6.49
35–47 D	7.85	9.22	11.63	24.77	27.46	26.85	14.13	14.09
FS	2.86	3.37	6.60	6.25	9.51	11.02	5.42	6.38
FS D-B	6.55	6.86	21.83	18.29	23.76	28.80	12.37	13.75
FS D-m	6.43	6.71	8.33	15.15	21.45	21.24	12.05	12.97
FS D-s	6.13	5.98	6.92	8.89	16.71	14.63	11.41	11.56
CD	0.014		0.011		1.130		0.003

Boddey et al. (1991) reported that in case of sugarcane, plant associated nitrogen fixation was due to N₂ fixing bacteria A. diazotrophicus. Similarly, James et al. (1994) demonstrated that A. diazotrophicus, an acid tolerant endophytic bacterium, fixes significant amount of nitrogen in sugarcane. Several works have confirmed nitrogen fixation by Azospirillum too, using not only conventional micro-Kjeldhal assay, but also the more definite method of isotopic enrichment involving ¹⁵N (Laxmi Kumari et al. 1976, Okon et al. 1976b, Barber et al. 1978, Scott & Scott 1978).

Biomass and yield

Biomass of roots, carpels, leaves and stem of each plant inoculated with parent of bacterial strains and their respective mutants was determined in both cotton varieties. Biomass of plants inoculated with mutants was significantly higher than the plants inoculated with their respective parent strains (Table III).

Table III Effect of parental strain/ mutants of Acetobacter diazotrophicus and Azospirillum brasilense on total biomass and yield in cotton genotypes.

Strains/mutants	Biomass (g)
	Root		Leaves		Stem		Carpel		Yield (g)
	HD-123	H-117	HD-123	H-117	HD-123	H-117	HD-123	H-117	HD-123	H-117
35–47	10.6	11.8	13.65	18.02	16.01	15.9	10.8	12.2	7.69	14.23
35–47D	12.8	14.3	18.22	18.55	20	19.4	13.7	13.6	25.35	22.79
FS	11.3	10.5	10.7	9.85	12.1	17.1	11.4	12.9	10.40	10.56
FS D-B	12.1	12.0	17.27	14.99	18.1	17.2	12.7	13.6	20.06	18.21
FS D-m	11.4	11.6	6.83	11.77	16.7	16.3	12.5	13.1	14.46	12.86
FS D-s	11.0	10.0	5.81	7.38	15.1	13.4	12.0	11.8	9.49	8.64
CD	0.261		0.001		0.121		0.072		2.24

The flower bud formation started on the 45th day. Plants inoculated with mutants showed early and more flower buds than plants inoculated with parent strains (Figure 1). There was a significantly higher yield from the plants inoculated with mutants as compared to plants inoculated with their respective parent strains. Even biomass and yield were also higher in plants inoculated with Acetobacter than the plants inoculated with Azospirillum. Thus, the present study revealed a positive correlation between nitrogen content and biomass and yield (lint + seed), and also nitrogenase activity is positively related to the nitrogen fixing efficiency.

Figure 1.  (A) Effect of Acetobacter diazotrophicus (35–47) and its azide resistant mutant (35–47D) on ball formation in American cotton (H-117). (B) Effect of Acetobacter diazotrophicus (35–47) and its azide resistant mutant (35–47D) on ball formation in Indian cotton (HD-123). (C) Effect of Azospirillum brasilenssse (FS) and its azide resistant mutants (FS D-B, FS D-M, FS D-S) on ball formation in American cotton (H-117). (D) Effect of Azospirillum brasilense (FS) and its azide resistant mutants (FS D-B, FS D-M, FS D-S) on ball formation in Indian cotton (HD-123).

Conclusion

These results indicated that azide resistant mutants are superior to their respective parent strains. The present work also resulted in material contributions in terms of endophytic and associative bacterial mutants that have capacity to influence plant growth in two contrasting genotypes of cotton. The wide use of synthetic fertilizers and chemicals has resulted in environmental degradation, decline of beneficial microorganisms and accumulation of chemical residues in the food web. For a sustainable agriculture, the use of biologically derived fertilizers and plant protection agents would be an ecologically sound and economically viable alternative. Thus, this supplementary study and further investigations will help to develop an integrated strategy for genetically manipulate bioinoculants and tailor crop genotypes for harnessing the genetic potential and favorable interaction among microbial and plant communities and enhance sustainable land use. The knowledge of biological nitrogen fixation in non-leguminous plants becomes one of the largest challenges in modern times, since it represents the more important alimentary base of the population, mainly in developing countries like India.

It is my privilege to express my gratefulness to the learned members of my advisory committee whose scholarly advice, for their critical suggestions and help to me in my research work. With sincerity and immense pleasure, I express my heartfelt gratitude to Dr Kamlesh Asotra, University of California, for his immense interest, sagacious guidance, intellectual stimulation and going through this manuscript. I express my special debt to CCS Haryana Agricultural University, Hisar, for awarding me a merit scholarship throughout my research work.