Abstract: Ten Azotobacter cultures were isolated from the maize rhizosphere and their auxin producing ability was measured colourimetrically. The auxin production by three efficient Azotobacter cultures (Z1, Z3, Z4) was also measured in the presence of filter sterilized L-tryptophan (at 10–3, 10–4 and 10–5 M). Azotobacter culture Z4 gave relatively higher auxin production and was selected for further experiments. Azotobacter inoculation in combination with 10–4 M L-TRP gave maximum length and weight of maize roots, which was 117 and 60 percent higher than control, respectively. Leonard Jar experiments were conducted to study the response of shoot growth to Azotobacter inoculation and L-TRP application at 10–4 M separately and in combination with each other. Results showed that maximum length and weight of shoots were recorded by applying Azotobacter in combination with 10–4 M L-TRP which was 45.3 and 36.5 percent higher than control, respectively. The possible mechanisms of action are discussed.
Introduction
It has been established and is now well accepted that normal plant growth and development throughout ontogeny is controlled by these compounds produced by the plant itself (Davies, 1987). However, plants may not have the capacity to synthesize sufficient endogenous plant hormones for optimal growth and development under sub-optimal growth and environmental conditions. Exogenously supplied plant hormones may affect plant growth by changing the balance of endogenous levels of hormones, allowing a modification of growth and development in desired direction and to the desired extent (Nickell, 1982).
Another potential and economical source of these phytohormones is the soil microbiota. A vast majority of soil microorganisms release these compounds (Frankenberger and Arshad, 1995; Arshad and Frankenberger, 1997). Studies have shown that microbial production of phytohormones can be increased several folds by providing their suitable precursors. These precursors may provide a continuous source of active substances due to the activities of rhizosphere microbiota for plant uptake and affect the plant growth because of the intimate contact between rhizosphere microbiota and plant roots which is better than one time application of synthetic compounds (Arshad and Frankenberger, 1990).
Many studies have shown the ability of inocula to produce plant hormones as one of the most plausible explanations for microbe-plant interactions (Hussain et al. 1987; Arshad and Frankenberger, 1991). The availability of suitable precursor is one of the primary factors affecting microbial secretion of these secondary metabolites. The exogenous application of precursors resulted in increasing the magnitude of phytohormone production in culture and soil by several folds (Frankenberger and Arshad, 1995).
L-Tryptophan (L-TRP) is considered an efficient physiological precursor of auxins in higher plants as well as for microbial biosynthesis of auxins (Arshad and Frankenberger, 1991). Frankenberger et al. (1990) reported the physiological response of radish (Raphanus sativus) to L-TRP applied to soil under optimal nutritional conditions. They observed a significant positive effect of L-TRP on growth parameters of radish when applied at low concentration at the seedling stage.
Zahir et al. (1997b) conducted a pot experiment to evaluate the effect of an auxin precursor L-TRP and Azotobacter inoculation on potato yield under fertilized conditions. They reported that Azotobacter inoculation when supplemented with L-TRP was more effective than their application alone in increasing tubers and straw yield (up to 69.9 and 47.8 percent, respectively) in comparison with control. Khalid et al. (1999) also reported similar results from a field experiment on wheat crop. According to them, combined application of Azotobacter and L-TRP increased the grain yield, straw yield and 1000-grain weight by 21.3, 20.7 and 6.4 percent, respectively, compared with untreated control.
Materials and Methods
A series of Plate and Leonard Jar experiments were conducted in a controlled temperature Growth Room (at 28 ± 1°C) under axenic conditions to study the effect of Azotobacter and L-TRP on growth of maize seedlings.
isolation of Azotobacter: Azotobacter cultures were isolated from maize rhizosphere by dilution plate technique using modified mannitol agar medium (Society of American Bacteriologists, 1957). Ten fast growing colonies were isolated, purified and numbered as Z1, Z2----Z10.
Measurement of auxin production: Sterilized modified mannitol broth (25 mL) taken in glass tubes was inoculated with ten Azotobacter cultures and incubated at 28 ± 1°C for 24 hours with occasional shaking. The contents of the tubes were filtered through whatman filter paper No.2 before measuring auxin production as indole acetic acid (IAA) equivalents. While measuring IAA equivalents, 3 mL of filtrate was taken in test tubes and 2 mL of Salkowski reagent (2 mL 0.5 M FeCI3 + 98 mL 35 percent HCIO4) was added to it. The mixture in the tubes was allowed to stand for 30 minutes for colour development. Intensity of the colour was measured at 535 nm by using spectronic -20. Similarly, colour was also developed in standard solutions of IAA and a standard curve was drawn by measuring the intensity of this colour (Sarwar et al., 1992). The auxin production by three efficient auxin producing Azotobacter cultures was also measured in the presence of filter sterilized L-tryptophan (5 mL of 5 percent solution) using above mentioned procedure. Azotobacter (Z4) culture giving best auxin production was selected for plate experiments.
Preparation of inoculum: Sterilized modified mannitol broth taken in conical flasks (250 mL flask) was inoculated with Azotobacter culture Z4 and incubated at 28±1°C for 4 days with occasional shaking (4-5 times a day). Fresh inoculum was prepared for each experiment.
Plate Experiments: Two plate experiments were conducted to study the effect of Azotobacter inoculation on the growth of maize roots both in the presence (10–3, 10–4 and 10–5 M) and absence of L-tryptophan. Inoculated seeds (dipping in broth for half an hour) were grown (4 seeds plate–1) for ten days on sterilized moist filter paper. L-Tryptophan was applied at 2 mL plate–1. The precursor-inoculum combination giving best results was selected for subsequent Leonard jar experiments. Data regarding the length and weight of maize roots was recorded.
Leonard Jar Experiments: Plate experiments were repeated in Leonard jar to study the effect of Azotobacter inoculation with and without L-tryptophan on the growth of maize shoots. In these experiments, Hoagland solution was taken in glass jars and sand in plastic glasses with a wick passed through glass bottom and dipped in the treatment solution. The apparatus was autoclaved prior to the transfer of germinated seeds. L-Tryptophan and broth inocula of selected Azotobacter culture were applied at 5 ml, respectiiitily, in sand. The duration of experiment was 2 weeks. Plate and Leonard jar experiments were conducted in growth room at 28 ±1°C. Data regarding the length and weight of maize shoots were recorded.
Statistical procedures were applied to analyse the data (Steel and Torrie, 1980) using completely randomized design and means were compared by Duncan's Multiple Range Test (Duncan, 1955).
Results
Effect of microbially derived auxins on the growth of maize seedlings was studied. The results are presented as below.
Microbial (Azotobacter) production of auxins: Data (Fig. 1) revealed auxin production by ten Azotobacter culture which ranged from 1.5 to 3.1 mg L–1 . Azotobacter culture Z4 showed the highest auxin production (3.1 mg L–1). Auxin production by all other Azotobacter cultures was followed in descending order by Z1, Z3, Z5, Z7, Z2, Z10, Z6, Z9 and Z8, respectively. Three efficient auxin producers (Z4, Z1 and Z3) were selected for further experimentation.
The selected Azotobacter cultures were used for measuring substrate (L-TRP) dependent auxin production. Data (Fig. 2) revealed that supplementation with L-TRP at 10–3 M resulted in maximum increase (171 percent) in auxin production by Azotobacter culture Z4, while Z1 gave maximum auxin production at L-TRP concentration of 10–4 M, which was 148 percent greater than the respective control. Being the most efficient auxin producer, Azotobacter culture Z4 was selected for further experimentation.
| Table 1: | Effect of Azotobacter and L-tryptophan on growth of maize (var. Golden) roots under normal conditions (Plate experiments) (Average of 4 replicates) |
| Means sharing the same letter(s) do not differ significantly at p = 0.05 | |
| Table 2: | Effect of Azotobacter and L-tryptophan on shoot growth of maize (var. Golden) under normal conditions (Leonard jar experiment) (Average of 4 replicates) |
| Means sharing the same letter (s) do not differ significantly at 1)=0.05 | |
Root growth: Data (Table 1) revealed that Azotobacter inoculation significantly increased the root length by 68.3 percent in experiment 2, while had non-significant effect in experiment 1. Similarly, all levels of L-TRP significantly increased the root length in case of experiment 2 and highest root length (30.0 cm) was observed under the treatment of 10–5 M L-TRP, which was 82.9 percent higher than control. All the Combinations of Azotobacter and L-TRP significantly increased the root length in both of the experiments and maximum increases (117 and 107.3 percent, respectively) in root length were observed by applying Azotobacter plus 10–4 ML-TRP.
In case of root weight, similar kind of response was observed and root weight was increased (by 33.8 percent) significantly by Azotobacter inoculation iri experiment-2, while it had non-significant effect in experiment-1. L-Tryptophpn application at 10–3, 10–4 and 10–5 M significantly increased the root weight and maximum root weights were observed by applying 10–5 M L-TRP which were 0.89 and 1.06 g in experiment-1 and 2, respectively.
| Fig. 1: | Measurement of auxin production (IAA equivalents) by different Azotobacter cultures |
Combined application of Azotobacter and L-TRP at 10–3, 10–4 and 10–5 M also significantly increased root weight and maximum increases (60 and 110.3 percent) were observed by applying Azotobacter plus 10–4 M L-TRP in both experiments, respectively.
Shoot growth: It is evident from data (Table 2) that Azotobacter inoculation and L-TRP application at 10–4 M significantly increased the shoot length by 25.0 and 22.2 percent, respectively, compared to uninoculated control.
| Fig. 2: | Effect of L-TRP on auxin production (IAA equivalents) by three Azotobacter cultures |
Similarly, compared to uninoculated control. Similarly, combined application of Azotobacter and 10–4 M L-TRP also enhanced the shoot length significantly by 45.3 percent compared to control.
In case of shoot weight, similar kind of response was observed and increased (by 10.6 and 19.4 percent) significantly by Azotobacter inoculation and L-TRP application at 10–4 M, respectively. Combined application of Azotobacter and 10–4 M L-TRP also promoted shoot weight by 36.5 percent significantly compared to uninoculated/untreated control.
Discussion
The current studies revealed that inoculation with Azotobacter alone or, in combination with L-TRP (an auxin precursor) had significantly positive effects on growth of maize seedlings under axenic conditions. However, the combined application of inoculation and precursor was more effective in improving growth of maize roots and shoots than their alone application.
The beneficial effects of Azotobacter on growth and yield of plants have been explained in terms of various mechanisms such as N2-fixation, production of plant growth regulating substances, alteration in microbial balance of soil, improved mineral uptake, production of siderophores and suppression of pathogenic microorganisms (Arshad and Frankenberger, 1993; Zahir et al., 1997a). The ability of Azotobacter to produce auxins had been reported by Mahmoud et al. (1984) and is considered the most plausible mechanisms of action to explain the beneficial effects of Azotobacter. As L-TRP is considered the most common precursor of auxins, so, supplementation of L-TRP increased the production of auxins by Azotobacter (Frankenberger and Arshad, 1995). The positive effects of L-TRP observed on the growth of maize may be due to direct uptake of L-TRP by plant roots with subsequent catabolism to auxins within the plant tissues. Zelena et al. (1988) reported similar results while working on maize. In our studies, higher plant growth in response to combined application of Azotobacter and L-TRP than their separate application could be attributed to microbially derived metabolites such as auxins. Similar to our findings, Khalid et al. (1999) also reported combined application of Azotobacter and 10–4 M L-TRP to be more effective than their separate application in a field trial on wheat. However, more intensive work is still needed to further verify the validity of this approach.