Efficacy of Azotobacter chroococcum in Rooting and Growth of Eucalyptus camaldulensis Stem Cuttings

MATERIALS AND METHODS

Isolation of A. chroococcum: This study was conducted at Model Nursery of Institute of Forest Genetics and Tree Breeding, Coimbatore, Tamilnadu (India) from September 2009 to February 2010. A. chroococcum was isolated from the rhizoplane of 7 years old E. camaldulensis growing in Coimbatore. Root segments with adhered substrate were placed in centrifuge tube and centrifuged for 5 min at 1000 rpm. Then the supernatant was collected and made up to 100 mL^-1 with sterile distilled water. One mL of the diluted supernatant was placed in Nitrogen free Jensen’s medium (sucrose 20 g, di-potassium hydrogen phosphate 1 g, magnesium sulfate 0.5 g, sodium chloride 0.5 g, ferrous sulfate 0.1 g, sodium molybdate 0.005, agar 20 g, for 1 liter, pH 6.9). After 5 days of inoculation the bacterial colonies appeared on the media. The morphological characters were carefully observed and recorded. Further confirmation of A. chroococcum was bio chemically characterized by Indole, Methyl Red-Voges Proskaur test, carbohydrate fermentation, oxidase and urease tests as per the standard protocols (Cappuccino and Sherman, 1992). The culture was deposited in the bacterial culture collection of IFGTB and numbered as AB I 08.

Experimental design: Stem cuttings of E. camaldulensis with uniform height (4 cm) and healthy leaf and axillary bud were collected from Karunya Nagar, Coimbatore, India and preserved in ice box for 1 h. The cuttings were dipped in 1% Bavistin (Carbendazim) fungicide to avoid fungal infection. Then A. chroococcum inoculum suspension at 5x10⁷ cfu mL^-1 (5 and 10 mL) was applied to the stem cuttings at the rate of 5 and 10 mL placed in root trainers (100 cc) containing rooting substrate (vermiculite). Untreated cuttings and Indole Butyric Acid (IBA) treated (1000 ppm with talcum powder) were also maintained to compare the effect of A. chroococcum on rooting of E. camaldulensis. Each treatment was replicated at 15 times and arranged in a randomized block design. The cuttings were maintained for 30 days in mist chamber (33°C, 65%) and no nutrients were supplied.

Treatments: There were four treatments including an untreated control as follows:

Harvest and assessment of stem cuttings: After 30 days of treatment the stem cuttings were harvested and the assessments of root number and length, leaf number and bio mass were made. The percent root initiation was used as a measure to asses the influence of A. chroococcum and IBA on the rooting of E. camaldulensis cuttings and was calculated using the formula:

Estimation of A. chroococcum for Indole-Acetic Acid (IAA) production: A. chroococcum was estimated for IAA production by the method of Loper and Schroth (1986). The presence of IAA in A. chroococcum culture was determined by development of pink colour and the concentration was measured in UV spectrophotometer at 530 nm.

Statistical analysis: Each measured variable in the nursery experiment were subjected to analysis of variance and means were separated using Duncan’s multiple range test using SPSS (ver. 10) software at the significant level of p<0.05.

RESULTS

Growth and rooting of E. camaldulensis stem cuttings: E. camaldulensis stem cuttings inoculated with A. chroococcum showed significantly increased higher growth and bio mass rather than IBA treated and control stem cuttings. Stem cuttings inoculated with 10 mL broth of A. chroococcum showed two to three fold increase of (significant at p<0.05) higher shoot length, root length, No. of lateral roots and bio mass than IBA treated cuttings. The R/S ratio was also found significantly (p<0.05) lower in A. chroococum (0.754) inoculated stem cuttings than IBA treated cuttings (0.929) (Table 1).

Percent root initiation: Significantly (p<0.05) 90% root initiation was obtained in 10 mL broth of A. chroococcum inoculated stem cuttings whereas 5 mL broth of A. chroococcum induced 60% of root initiation and IBA treated stem cuttings showed only 28% of root initiation (Fig. 1).

IAA production of A. chroococum: IAA production was found in A. chroococcum broth with tryptophan addition. Higher concentrations of IAA occurred with increasing concentrations of tryptophan (Fig. 2).

Table 1:	Growth, rooting and biomass of E. camaldulensis stem cuttings inoculated with A. chroococum (mean of 15 replicates)
Means followed by same letters are significantly not different at p<0.05 according to Duncan’s Multiple Range Test

Fig. 1:	Percent root initiation of E. camaldulensis stem cutting inoculated wr A. chroococum (mean of 10 replicates) T1 A. Chroococum (5 mL), T2 A. Chroococum (10 mL), means followed by same letters are not significantly not different at p<0.05 according to Duncans multiple range test

Fig. 2:	IAA production of A. chroococum (mean of ten isolates)

DISCUSSION

Microbes always play in nutrient management of soil to improve soil fertility (Vikram and Hamzehzarghani, 2008). The application of the N₂ fixing bacteria A. chroococcum was first proposed in the mid 1970s as a new approach to provide fixed N to reduce fertilizer requirement and to increase yield (Dart, 1976). In present study A. chroococcum induced better rooting and growth of E. camaldulensis stem cuttings. This is in accordance with earlier reports where increased plant growth and bio mass have reported in response of N₂ fixing bacterial inoculation (Akbari et al., 2007; Fallik et al., 1998). This study also confirms the observations of Karthikeyan and Suryaprakash (2008) where inoculation of N₂ fixing bacteria Azospirillum brasilense resulted in better growth of E. camaldulensis seedlings. The stimulation of rooting in E. camaldulensis stem cuttings by A. chroococcum suggests its ability to produce plant hormones that could stimulate root initiation. The root exudates also promote the growth of microbe in the inert media which helps to survive the bacteria as reported in previous study (Kumar et al., 2007). Further In an earlier study it was indicated that about 80% of the bacteria present in soil are able to produce IAA (Glick et al., 1999). The production of IAA and important hormone for rooting by A. chroococcum was confirmed in this study. It was reported that IAA is the important hormone which promote the rooting in Vitellaria paradoxa cuttings (Yeboah et al., 2009) and Balanites aegyptiaca cuttings (Mostafa and Alhamd, 2011). Earlier studies have shown that Azotobacter species are able to produce IAA, GA and Cytokinin like substances both under culture conditions and in the plant rhizosphere (Brown, 1976; Jain and Patriquin, 1985). Inoculation of A. chroococum stimulated rooting in E. camaldulensis stem cuttings better than IBA. This suggests that continuous release of small quantities of IAA might enhance root initiation better than a single application of root hormone. In present study sufficient amount (7.8 μg mL^-1) of IAA to promote root initiation and elongation was produced even without supplement of tryptophan. This is evidenced by the significant variations observed in percentage of root initiation in E. camaldulensis cuttings with inoculation of A. chroococcum. Similar responses to A. chroococcum inoculation have been reported for mulberry (Das et al., 1990) and Bamboo (Dhamangaonkar and Misra, 2009) and Ocimum sanctum (Vinutha, 2005). Therefore, this study emphasized the need of rooting hormone synthesizing inoculants such as A. chroococcum for the propagation of E. camaldulensis and to reduce the usage of costlier synthetic root hormones and chemical fertilizers.

CONCLUSION

Studies on E. camaldulensis cuttings with inoculation of A. chroococcum do indicate that it can reduce fertilizer requirement in plant production. For quality cuttings production of commercial tree crops like E. camaldulensis consideration should be given to N₂ fixing microbes like A. chroococcum rather than costlier synthetic root hormones and chemical fertilizers. Moreover, this microbial inoculation techniques is cheap and easy to handle, could be adopted in clonal nurseries to improve the rate of growth and quality of E. camaldulensis cuttings without chemical fertilizers thus avoiding the associated costs.

ACKNOWLEDGMENT

The authors thanked to Indian Council of Forestry Research and Education, Dehradun, India for financial support for this study.