Root-enhancing properties of rhizospheric bacteria on Eucalyptus hybrid cuttings

Abstract

Worldwide, the demand for hardwood from commercial plantations is rising as the consumption of forest products increases. In Eucalyptus plantation forestry the formation of interspecific hybrids has driven the commercial forestry industry to produce a variety of different hybrids that are often deployed in marginal areas. Superior hybrid genotypes are deployed through the application of vegetative propagation and the rooting of cuttings. However, the cuttings of some hybrid genotypes demonstrate relatively low rooting percentages, which results in financial losses experienced by many commercial forestry nurseries. This study was thus undertaken to assess the potential of rhizospheric bacteria to improve the rooting capacity of two hybrids clones of E. grandis × E. nitens (GN 018B and GN 010). Methods: Firstly, the rhizospheric bacterial composition of Eucalyptus rhizospheric soils of different ages were characterised using fatty acid methyl ester (FAMEs) analyses. Thereafter, rhizospheric bacteria were isolated from Eucalyptus rhizospheres and characterised by sequencing the ≈1,300 base pair fragment of the 16S rRNA gene, after which the sequences were submitted to BLAST searches to identify the bacterial strains. Thereafter, the identified bacterial strains were tested for two important root promoting characteristics; the ability to produce indole-3-acetic acid and to solubilise phosphates. Bacterial inoculums were prepared and tested on cuttings of the two hybrids in the nursery. Four treatments were applied to the cuttings; the nursery standard, which acted as the control, two treatments prepared from the isolated rhizospheric bacteria and a commercial treatment containing a live fungus. Cutting survival, rooting, rooting architecture and growth were measured after eight weeks of growth. Results: This study revealed that rhizospheric microbial communities’ diversity evolved over time. Younger rhizospheres displayed significantly greater diversity when compared to the older rhizospheres. The presence of higher concentrations of saturated fatty acids in younger rhizospheres than in the older rhizospheres was indicative of aerobic conditions, while increased proportions of polyunsaturated fatty acids in the older rhizospheres indicated anaerobic conditions. The greater proportion of polyunsaturated fatty acids in the older rhizospheric soil samples, especially linoleic (C18:2ω6c), α-linolenic (C18:3ω3c), γ-linolenic (C18:3ω3c) fatty acids, as well as the unique γ-linolenic fatty acids, suggest growing establishment of fungi as a rhizosphere ages. Both Shannon and Simpson’s indexes confirmed that younger soils were more diverse. Thirty two rhizospheric bacterial strains were isolated and identified. These strains belonged to 12 unique species of eight different genera. Of the 12 rhizospheric bacterial species, seven demonstrated the ability to produce indole-3-acetic acid and to solubilise phosphates. These seven strains were used to prepare the inoculums to treat the cuttings of the two hybrids. Bacillus aryabhattai, Pseudomonas fluorescens, P. koreensis and P. putida were mixed into one rooting treatment, while Brevibacterium frigoritolerans, Burkholderia phytofirmans and Chryseobacterium rhizosphaerae were mixed into another treatment. Cuttings of both hybrid clones demonstrated relatively high survival rates and rooting percentages for all treatments, although the nursery standard treatment (indole-3-butyric acid) marginally outperformed all other treatments. The cutting growth responses of the two hybrids showed highly significant differences amongst the treatments (P < 0.0001). The rhizospheric rooting treatment comprising of the non-Pseudomonas-Bacillus bacteria, as well as fungus treatment, were closest in performance to the nursery standard, while the Pseudomonas-Bacillus treatment often showed lower values when compared to the other treatments. However, Pseudomonas-Bacillus bacterial treatment appeared to improve fibrosity of the rooting architecture of cuttings of one of the hybrid genotypes, which could be of value when rooted cuttings are planted out in plantations. A noteworthy outcome of this study was the finding that extensive genotypic differences existed between the two hybrid clones. Conclusions: The aim of this study was to test the application of rhizospheric bacteria as root stimulating agents in the cuttings of two Eucalyptus hybrid genotypes. The rhizospheric bacterial preparations used in this study did not enhance the number of rooted cuttings when compared to the nursery standard in a significant way, however, there were strong indications that the rhizospheric bacterial inoculums did promote fibrosity. These findings suggest further investigation into the formulation and application of potential rhizospheric bacterial preparations for rooting enhancement of Eucalyptus cuttings.