Elsevier

Soil Biology and Biochemistry

Volume 57, February 2013, Pages 436-443
Soil Biology and Biochemistry

Interactions between arbuscular mycorrhizal fungi, rhizobacteria, soil phosphorus and plant cytokinin deficiency change the root morphology, yield and quality of tobacco

https://doi.org/10.1016/j.soilbio.2012.09.024Get rights and content

Abstract

Arbuscular mycorrhizal fungi (AMF) and rhizobacteria (Pseudomonas fluorescens) have been targeted for plant growth promotion in order to reduce agrochemical inputs. However, the effects of their interaction on root morphology under different nutrient levels are unknown. Moreover, both soil microorganisms can alter the levels of plant hormonal regulators but no in vivo evidence is available for their interplay with cytokinin (CK) on root morphology. In a full-factorial greenhouse experiment we investigated the effects of interaction between Rhizophagus intraradices, P. fluorescens 8569r, phosphorus (P) amendment and plant CK deficiency on tobacco root morphology, shoot yield and quality. Our results suggest that reduced CK levels may be involved in plant signaling to stimulate AMF hyphal growth in the roots. In addition, we document that a bacteria isolated from the rhizosphere of a non-mycorrhizal plant can function as mycorrhizal helper bacteria, most likely via interplay with phytohormones. The two soil microorganisms, depending on the inoculum combination, P amendment and plant CK levels, modified tobacco root morphology. Our results suggest that the positive interactions between P. fluorescens and AMF depend on soil nutrient status and root hormonal balance. Both microorganism modified shoot yield and these effects seem to result from altered root morphology. Overall, our study support early conclusions that the classification of a soil microorganism as detrimental or beneficial should be based upon their net effects on the plant growth according to circumstances. We suggest this consideration to be extended to the effects of interaction between soil microorganisms on root morphology.

Highlights

► We studied intrinsic and extrinsic abiotic and biotic root morphology regulators. ► Plant cytokinin (CK) deficiency increased AMF hyphal root colonization. ► Pseudomonas fluorescens 8569r can function as mycorrhizal helper bacteria depending on CK. ► Interacting AMF, P. fluorescens 8569r, phosphorus and CK changed root morphology. ► Shoot yields were associated with altered root morphology.

Introduction

Modern agriculture relies on high input of agrochemicals which cause major environmental problems (Spiertz, 2010). Feeding an increasing human population and reducing the impacts on the environment urges for low input agricultural practices. Some argue that the solution can be found belowground in the rhizosphere (Gewin, 2010). There is still room for improvement by better exploring the genetic variability of roots to improve root morphology, their ability to forage or fixate nutrients or by manipulating soil microbes which improve the root function (Gewin, 2010).

Essentially, the root system is responsible for the acquisition of water and nutrients from the soil. Due to soil heterogeneity, a root morphology adapted to the soil environment is critical for plant survival and yield (Malamy, 2005). The morphological responses of roots to soil abiotic factors such as mineral nutrients have been well documented and reviewed (e.g. Forde and Lorenzo, 2001; Lopez-Bucio et al., 2003). However, our understanding of the effects of multiple biotic interactions belowground on root morphology is still in its infancy. This is particularly important when considering the development of microbial inoculations as a solution to improve plant growth and reduce chemical inputs.

Among the soil organisms targeted for plant growth promotion, the ubiquitous arbuscular mycorrhizal fungi (AMF) and Pseudomonas fluorescens have received considerable attention (e.g. Lugtenberg and Kamilova, 2009; Smith and Smith, 2011a). AMF can promote the growth of plants by enhancing the uptake of nutrients, in particular phosphorus (P) (Smith and Smith, 2011a). In addition, AMF can alter root morphology in most studies by inducing a more profusely root branching (Berta et al., 1993, 2005; Gamalero et al., 2002; Gamalero et al., 2004; Gutjahr et al., 2009; Olah et al., 2005). In contrast to AMF, changes in root morphology induced by P. fluorescens are not so often reported; most studies focus on the enhancement of root growth and length (Dhillion, 1992; Gamalero et al., 2003; Germida and Walley, 1996; Neeraj and Singh, 2011) with fewer showing positive or neutral effects on root surface area, volume and branching (Berta et al., 2005; Gamalero et al., 2002, 2004). To the extent of our knowledge, only one study has addressed the effects of interactions between P. fluorescens and AMF on root morphology, showing these to be either neutral or synergistic (Gamalero et al., 2004). However, the soil microorganisms are not always beneficial and the function of AMF and P. fluorescens may change depending on the edaphic conditions, such as nutrient availability and microbial densities (Alström, 1987; Gamalero et al., 2002; Hoeksema et al., 2010; Johnson et al., 1997). This raises the question if the effects of this biotic interaction on root morphology could be modified by abiotic factors such as fertilization, in particular with P.

Furthermore, the biotic and abiotic regulators of root morphology are both part of the responsive pathway of root morphogenesis (Malamy, 2005). Ultimately, it is the intrinsic pathways, i.e. the genetic and hormonal regulators, which modulate organogenesis and growth and determine the characteristic morphology of the root as induced by the responsive pathway (Malamy, 2005). Cytokinin (CK) is an important intrinsic, negative regulator of root growth and elongation and a systemic signaling of plant P starvation (Franco-Zorrilla et al., 2005; Werner et al., 2003, 2001). AMF colonization can alter the CK levels in the roots, irrespective of the improvement of P nutrition, which may result from indirect modification of plant hormonal production or directly by fungal CK synthesis in the root (Shaul-Keinan et al., 2002; Torelli et al., 2000). Moreover, it seems likely that CK may be involved in the AMF alteration of root morphology, but no experimental evidence exists supporting this mechanism. The enhancement of root length by P. fluorescens have been associated to bacterial synthesize of auxin (Gamalero et al., 2003, 2004), and the later is known to act in conjunction with CK to modulate root morphogenesis (Werner and Schmülling, 2009).

To further elucidate the relations between intrinsic and extrinsic abiotic and biotic regulators of root morphology, we have focused on the interaction among Rhizophagus intraradices, P. fluorescens, P amendment and plant CK levels, by comparing a tobacco wild type (WT) to a CK-deficient line. Furthermore, in agricultural systems the root efficiency in N uptake is of high importance for crop production (Spiertz, 2010). This is particularly important for tobacco, whose leaf yield and nicotine, an important chemical trait of tobacco quality, are strongly affected by N availability (Fritz et al., 2006; Ruiz et al., 2006; Russell et al., 1980; Steppuhn and Baldwin, 2007). Therefore, we evaluated as well the competence of root morphological changes induced by the biotic and abiotic interactions in affecting tobacco shoot yield and concentrations of N and nicotine in leaves.

Section snippets

Plant lines and soil microorganisms

As experimental plants we used two lines of tobacco (Nicotiana tabacum L. cv. Samsun NN), the WT and the 35S:CKX2 transgenic line. The latter was developed and reported by Werner et al. (2001). Shortly, the 35S:CKX2 line was constructed by cloning a gene encoding a protein with cytokinin oxidase (CKX) activity, the AtCKX2 gene from Arabidopsis thaliana. The gene was positioned in the tobacco WT under the control of a constitutive 35S promoter. The line 35S:CKX2 overexpress CKX and,

Microbial root colonization

At the end of the experiment, the plants treated with AMF inoculum had on average a low AMF root colonization (11% ± 0.33, Mean ± SE), varying from 2 to 28%. In the plants treated with the AMF mock solution no AMF root colonization was observed. To test the effects of the other factors (i.e. plant line, P amendment and P. fluorescens 8569r) on AMF root colonization, only the plants inoculated with AMF were considered. We detected a significant two-way interaction between plant line and

Discussion

In our experimental set up the context was important for the effects of AMF, P. fluorescens and their interaction on root morphology as well as on shoot yield and quality. This supports early conclusions that the classification of soil microorganisms as detrimental or beneficial, particularly in systems managed by humans, should be based upon their net effects on plants growth according to circumstances (Johnson et al., 1997; Nehl et al., 1997). Our study supports the extension of this

Conclusion

Our study has shown that CK deficiency can encourage AMF hyphal growth in the roots and that P. fluorescens isolated from the rhizosphere of a non-mycorrhizal plant can function as MHB, depending on plant CK levels. The two soil microorganisms, depending on the inoculum combination, P amendment and plant CK levels, modified tobacco root morphology. The directions of the interaction effects between AMF and P. fluorescens 8569r on root morphology seem to depend on the nutrient status in the

Acknowledgments

This research project was funded by the Center for International Cooperation (CIC) and the Dahlem Center for Plant Science (DCPS) of Freie Universität Berlin. We thank Prof. Thomas Schmülling and Dr. Eswar Ramireddy for providing us with the tobacco seeds, greenhouse facilities and expertise on cytokinin, Prof. Anke Steppuhn for the HPLC equipment, laboratory protocols and expertise on nicotine, and Prof. Matthias Rillig for the laboratory protocols and expertise on AMF.

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