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Besides the plant-fungus symbiosis in arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) plants, many endorhizal and rhizosphere bacteria (Root Associated Bacteria, or RAB) also enhance plant fitness, diversity, and coexistence among plants via bi- or tripartite interactions with plant hosts and mycorrhizal fungi. Assuming that bacterial associations are just as important for the obligate mycorrhizal plant family Orchidaceae, surprisingly little is known about the RAB associated with orchids. Herein, we first present the current, underwhelming state of RAB research including their interactions with fungi and the influence of holobionts on plant fitness. We then delineate the need for novel investigations specifically in orchid RAB ecology, and sketch out questions and hypotheses which, when addressed, will advance plant-microbial ecology. We specifically discuss the potential effects of beneficial RAB on orchids as: (1) Plant Growth Promoting Rhizobacteria (PGPR), (2) Mycorrhization Helper Bacteria (MHB), and (3) constituents of an orchid holobiont. We further posit that a hologenomic view should be considered as a framework for addressing co-evolution of the plant host, their obligate Orchid Mycorrhizal Fungi (OMF), and orchid RAB. We conclude by discussing implications of the suggested research for conservation of orchids, their microbial partners, and their collective habitats.

More than 95 % short roots of most terrestrial plants are colonized by mycorrhizal fungi as soon as they emerge in the upper soil profiles. The establishment of mycorrhizal association involves profound morphological and physiological changes in root and fungus. It is affected by other rhizospheric microorganisms, specifically by the bacteria. Bacteria may have developed mechanisms of selective interaction with surrounding microorganisms, with neutral or positive effects on mycorrhizal associations, but negative effect on root pathogens in general. Because of the beneficial effect of bacteria on mycorrhizae, the concept of Mycorrhization Helper Bacteria (MHB) was created. Five main actions of MHB on mycorrhizae were proposed: in the receptivity of root to the mycobiont, in root-fungus recognition, in fungal growth, in the modification of rhizospheric soil and in the germination of fungal propagules. MHB appear to develop a gradation of specificity for the mycobiont, but little or no specificity for the host plant in symbiosis. One of the main groups of MHB is the fluorescent Pseudomonas, well represented in diversity and cell density studies of mycorrhizal associations. This review covers the activity of MHB in the establishment of ectomycorrhizae, taking as model the effects of Pseudomonas sp. described in scientific literature.

A filamentous fungus was isolated from Tuber borchii Vitt. fruiting bodies, and it was identified as an Arthrinium phaeospermum (Corda) M.B. Ellis strain, an “endophyte” that forms various associations with healthy leaves, stems, and roots of plants. Molecular analysis confirmed the association of this filamentous fungus with the ascocarps of all collection sites in Salento, Apulia (South Italy). An in vitro symbiosis system between Cistus creticus L. and T. borchii was set up; A. phaeospermum appears to be able to promote mycorrhiza formation in Cistus seedlings, inducing primary root shortening and an increase of secondary roots, similar to the effect of Mycorrhization Helper Bacteria (MHB). Compartmented and uncompartmented bioassays were carried out to investigate the effects of exudates/volatiles released by the truffle-hosted fungus on root architecture; the results showed root shortening in compartmented bioassay suggesting that volatiles released by the fungus alone are sufficient to alter root morphology in early phase of interaction before the mycorrhiza formation. The first evidence for an influence of a truffle-hosted fungus on ectomycorrhizal symbiosis establishment is reported.

Abstract Mycorrhization helper bacteria, Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, were used to study the importance of bacterial inoculum dose and bacterial derived soluble and volatile metabolites localization for enhancing mycorrhiza formation in the Pinus sylvestris-Lactarius rufus symbiosis, using a laboratory based microcosm. EJP73 and EJP67 produced different responses in relation to the inoculum dose; EJP73 significantly enhanced mycorrhiza formation to the same degree at all doses tested (105, 107, 109 and 1010 CFU mL−1), whereas, EJP67 only stimulated mycorrhiza formation within a narrow range of inoculum densities (107 and 109 CFU mL−1). The importance of soluble bacterial metabolites was assessed by applying spent broth derived from exponential and stationary phase bacterial cultures to microcosms. No spent broth enhanced mycorrhiza formation over the control. As EJP73 produced the helper effect over a wide range of inoculum doses, this bacterium was chosen for further study. Physical separation of EJP73 from the fungal and plant symbiosis partners was carried out, in order to determine the contribution of constitutively produced bacterial volatile metabolites to the mycorrhization helper bacteria effect. When EJP73 was physically separated from the symbiosis, it had a significant negative effect on mycorrhiza formation. These results suggest that close proximity, or indeed cell contact, is required for the helper effect. Therefore, fluorescent in situ hybridization in conjunction with cryosectioning was used to determine the localization of EJP73 in mycorrhizal tissue. The cells were found to occur as rows or clusters (∼10 cells) within the mycorrhizal mantle, both at the root tip and along the length of the mycorrhizal short roots.

Mycorrhization helper bacteria (MHB), isolated from phylogenetically distinct ectomycorrhizal symbioses involving Lactarius rufus, Laccaria bicolor or Suillus luteus, were tested for fungus specificity to enhance L. rufus–Pinus sylvestris or L. bicolor–P. sylvestris mycorrhiza formation. As MHB isolated from the L. rufus and S. luteus mycorrhiza were originally characterised using a microcosm system, we assessed their ability to enhance mycorrhiza formation in a glasshouse system in order to determine the extent to which MHB are system-specific. Paenibacillus sp. EJP73, an MHB for L. rufus in the microcosm, significantly enhanced L. bicolor mycorrhiza formation in the glasshouse, demonstrating that the MHB effect of this bacterium is neither fungus-specific nor limited to the original experimental system. Although the five MHB strains studied were unable to significantly enhance L. rufus mycorrhiza formation, two of them did have a significant effect on dichotomous short root branching by L. rufus. The effect was specific to Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, the two strains isolated from L. rufus mycorrhiza, and was not associated with auxin production. Altered mycorrhiza architecture rather than absolute number of mycorrhizal roots may be an important previously overlooked parameter for defining MHB effects.

The development of ectomycorrhizas on inoculated eucalypt seedlings in commercial nurseries is often slow so that only a small percentage of roots are mycorrhizal at the time of outplanting. If mycorrhizal formation could be enhanced by co-inoculation with bacteria which promote rapid root colonisation by specific ectomycorrhizal fungi, as demonstrated by certain bacteria in the Douglas fir-Laccaria bicolor association, this would be of advantage to the eucalypt forest industry. Two bacterial isolates with a demonstrated Mycorrhization Helper Bacteria (MHB) effect on ectomycorrhiza formation between Pseudotsuga menziesii and Laccaría bicolor (S238), and seven Western Australian bacterial isolates from Laccaría fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaría spp. with Eucalyptus diversicolor seedlings.Mycorrhizal formation by L. fraterna (E710) as measured by percentage infected root tips, increased significantly (p < 0.05) by up to 296% in treatments coinoculated with MHB isolates from France (Pseudomonas fluorescens Bbc6 or Bacillus subtilis MB3), or indigenous isolates (Bacillus sp. Elf28 or a pseudomonad Elf29). In treatments coinoculated with L. laccata (E766) and the MHB isolate P. fluorescens (Bbc6) mycorrhizal development was significantly inhibited (p < 0.05). A significant Plant Growth Promoting Rhizobacteria (PGPR) effect was observed where the mean shoot d.w. of seedlings inoculated only with an unidentified bacterium (Elf21), was 49% greater than the mean of uninoculated controls (-fungus, -bacterium). Mean shoot d.w. of seedlings coinoculated with L.bicolor (S-238), which did not form ectomycorrhizas with E. diversicolor, and an unidentified bacterium (Slf 14) or Bacillus sp. (Elf28) were significantly higher than uninoculated seedlings or seedlings inoculated with L. bicolor (S-238) alone. This is the first time that an MHB effect has been demonstrated in a eucalypt-ectomycorrhizal fungus association. These organisms have the potential to improve ectomycorrhizal development on eucalypts under nursery conditions and this is particularly important for fast growing eucalypt species where the retention time of seedlings in the nursery is of short duration (2-3 months).

By dint of the development of agroecological practices and organic farming, stakeholders are becoming more and more aware of the importance of soil life and banning a growing number of pesticide molecules, promoting the use of plant bio-stimulants. To justify and promote the use of microbes in agroecological practices and sustainable agriculture, a number of functions or services often are invoked: (i) soil health, (ii) plant growth promotion, (iii) biocontrol, (iv) nutrient acquiring, (v) soil carbon storage, etc. In this paper, a review and a hierarchical classification of plant fungal partners according to their ecosystemic potential with regard to the available technologies aiming at field uses will be discussed with a particular focus on interactive microbial associations and functions such as Mycorrhiza Helper Bacteria (MHB) and nurse plants.