Arbuscular mycorrhizal fungi (AMF) and phosphate solubilizing Pseudomonas bacteria (PSB) could potentially interact synergistically because PSB solubilize phosphate into a form that AMF can absorb and transport to the plant. However, very little is known about the interactions between these two groups of microorganisms and how they influence the growth of each other. We tested whether different strains of bacteria, that have the capacity to solubilize phosphate, are able to grow along AMF hyphae and differentially influence the growth of AMF both outside the roots of carrot in in vitro conditions and inside the roots of potato in the presence of a microbial community. We found strong effects of AMF on the growth of the different bacterial strains. Different bacterial strains also had very strong effects on the growth of AMF extraradical hyphae outside the roots of carrot and on colonization of potato roots by AMF. The differential effects on colonization occurred in the presence of a microbial community. Our results show that these two important groups of rhizosphere microorganisms indeed interact with each other. Such interactions could potentially lead to synergistic effects between the two groups but this could depend on whether the bacteria truly solubilize phosphate in the rhizosphere in the presence of microbial communities.

Introduction

Arbuscular mycorrhizal fungi (AMF) are present in nearly all soils, forming associations with roots of approximately 80% of plant species [1]. In exchange for photosynthates, AMF provide phosphate (P) and other nutrients to their plant hosts by producing hyphae that grow out from roots, effectively increasing the soil volume from which minerals are acquired [1]. The capacity of AMF to transport P to the plant, which in some cases adds up to 70% of total P plant uptake, is well known [1]. However, AMF can only exploit soluble P sources and much P in the soil is in an insoluble form.

The rhizosphere is regarded as a hotspot for microbial activity and recent studies indicate that this is also the case for the mycorrhizosphere where soil bacteria may attach to extraradical AMF hyphae [2]. The effect of arbuscular mycorrhizal fungal colonisation on the soil microbial community has been little studied. Changes in soil bacterial community composition due to the presence of AMF have been described, both in vivo and in vitro [3, 4]. Among soil bacteria in the rhizosphere, those with the capacity to solubilize P are highly relevant when studying AMF interactions because these bacteria could potentially make available more soluble P for absorption by AMF hyphae.

Phosphate solubilizing bacteria (PSB) are free-living soil microorganisms that are present in most soils [5]. In in vitro conditions, they have the potential to improve availability of P to the plant by solubilizing organic and inorganic P, through the action of synthetized phosphatases, by lowering the pH of the soil, and/or chelating P from Al3+, Fe3+, Fe2+ and Ca2+ with the help of organic acids [5, 6]. The most widely used method to initially select microorganisms with P solubilization capacity in vitro is the use of tri-calcium phosphate, although the predictability of this assay for P solubilization in soil conditions is very limited (recently reviewed in [7]).

Many soil bacteria, including species of Pseudomonas, Azotobacter, Burkholderia, Bacillus and Rhizobium have been shown to have the capacity to solubilize poorly available P [6, 8, 9]. In particular, Pseudomonas spp., are known to colonize the rhizosphere, solubilize P and can exhibit additional plant growth promoting characteristics such as plant growth stimulation and the production of metabolites that have anti-microbial activity [10, 11].

However, the interactions between PSBs and AMF are poorly understood and the approach to using both these microbial groups for applications in agriculture is often naive because of variation in soil abiotic and biotic environments in which these organisms have often not been tested [12, 13]. Moreover, very often, only single strains of these microbial groups have been shown in laboratory or greenhouse conditions to have the capacity to solubilize P or to improve plant P acquisition. Indeed, higher plant P uptake capacity has previously been reported when plants are co-inoculated in greenhouse conditions, with AMF and PSB [14, 15]. These bacteria probably improve the availability of P, which can subsequently be efficiently absorbed by AMF hyphae [14, 16]. Thus, on the basis of results, mostly from artificial experiments conducted in sterilized soil, AMF and PSB are thought to act synergistically. Recent evidence also points, not only to synergistic effects between AMF and PSB but also to cooperation between these organisms [17]. However, most of the beneficial effects of AMF are observed in experiments conducted in sterile soil [13]. In reality, plants naturally become colonized by the local AMF community. A more realistic test of their potential is whether adding AMF inoculum and PSBs to unsterilized soil will give a growth benefit to the plant. Such tests are rarely performed. Isolated beneficial microbes are then used in field applications, where the bacteria and fungi encounter both diverse soil environments and diverse microbial communities, including existing diverse populations of both PSBs and AMF. It is perhaps unsurprising; therefore, that application of both AMF and PSBs in agriculture has had very variable success [12].

Given that both AMF and PSB must have co-existed in the rhizosphere for millions of years, many possible interactions could have evolved between them. Yet the interaction between AMF and PSB is not well understood. Firstly, in the mycorrhizosphere, the soil zone influenced by both the roots and the mycorrhizal fungi [18], AMF exudates create an environment that can influence bacterial growth [2, 4, 19]. Attachment of bacteria, with P solubilizing capacity, to extraradical AMF hyphae, could ensure that P solubilizing activities of the bacteria would be located in the zone where they can be most beneficial in allowing the fungi access to additional soluble P. At the same time, attachment to the AMF hyphae might provide bacteria with a route to efficiently access the mycorrhizosphere [20]. Some soil bacteria have been shown to attach both to vital and non-vital AMF hyphae in in vitro conditions [2]. However, none of the bacteria in that study were assessed for their P solubilizing capacity. It is unknown whether any bacteria with phosphate solubilizing capacity have the ability to attach to AMF extraradical hyphae [21]. Of those PSB that might associate with AMF hyphae, it is unknown whether these bacteria might influence either the growth of AMF inside the roots or of AMF hyphae outside the roots. A positive effect of PSB on extraradical AMF hyphal growth could help PSB to access new areas of the mycorhizosphere and increase access by AMF hyphae to new sources of solubilized P. Thirdly, populations of PSB are diverse in the soil [6, 22–25] and it is unknown whether there is variation among strains in the effects of PSB on AMF.

The aims of this study were to test: 1. To test whether different Pseudomonas spp strains, which have previously been shown to be capable of solubilizing P in in vitro conditions, differ in their ability to grow along AMF hyphae; 2. Whether the bacterial strains differentially influence the growth of the fungus outside the roots and in the absence of a microbial community and whether P solubilized by the bacteria can be transported by the fungus to the plant root; 3. Whether there is variation in the capacity of the bacterial strains to influence colonization by mycorrhizal fungi inside the roots and in the presence of a microbial community in a non-sterilized Colombian Andisols. We used ten Pseudomonas spp. strains, from Colombian Andisols that were previously isolated [26] and that were characterized as PSBs in this study. Here we define PSB as bacteria that have been shown to solubilize tri-calcium phosphate in in vitro conditions. Thus, the bacteria we consider possess the metabolic capability to solubilize phosphate. This, however, does not mean that these bacteria would indeed solubilize P in a variety of different soils and in the presence of a potentially competing microbial community. We focussed on PSB originating from Colombian Andisols because these soils are important for potato production but characterized by very high P retention due to acidic conditions (pH <5.5). We used the in vitro-produced AMF Rhizophagus irregularis because it has been shown to improve plant growth in field conditions in non-sterilized tropical acidic soils and has a global distribution [27].