Mycorrhization Helper Bacteria Research Articles

Microbial communities of ascocarps and soils in a natural habitat of Tuber indicum.

Truffles are the fruiting bodies of hypogeous fungi in the genus Tuber. Some truffle species usually grow in an area devoid of vegetation, called brûlé, but limited knowledge is available on the microbial composition and structure of them. Here, we investigated the bacterial and fungal communities of Tuber indicum ascocarps and soils inside and outside a characteristic brûlé from a poplar plantation with no truffle production history in northeastern China using a high-throughput sequencing approach. A predominance of members of the bacterial phylum Proteobacteria was observed in all samples. Members of Bacillus were the main genera in the ascocarps, while members of Lysobacter and unidentified Acidobacteria were more abundant in the soil. In addition, members of Gibberella, Fusarium, and Absidia were the dominant fungi in the ascocarps, while members of Tuber were enriched in the ascocarps and soils inside the brûlé. Some mycorrhization helper bacteria (Rhizobium) and ectomycorrhiza-associated bacteria (Lysobacter) were detected, indicating their potential roles in the complex development of underground fruiting bodies and brûlé formation. These findings may contribute to the protection and cultivation of truffles.

Orchid Root Associated Bacteria: Linchpins or Accessories?

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.

English

Stevia rebaudiana Bert. is a non-caloric sweetener plant of medicinal and commercial values. This study was performed to micropropagate Stevia and improve its ability to withstand the sudden shocks of the environmental changes during acclimatization by using endomycorrhizae and/or Pseudomonas fluorescens. In vitro establishment of Stevia was superior by culturing shoot tips and nodal segments of mature plants on Murashige and Skoog medium supplemented with 0.5 mg/l 6-benzyl adenine and 1 mg/l β-naphthalene acetic acid, and nodal segments gave better response when compared to shoot tips. While 6-benzyl adenine alone at 1 mg/l was found to be the most promising concentration for shoots multiplication for four successive subculturing. The best rooting response was obtained on half strength Murashige and Skoog medium supplemented with 1 mg/l β-naphthalene acetic acid. Adding 8 ml/l mycorrhizal root extract gave 100% rooting with the highest mean number and length of roots and shoot height. Inoculation of plantlets with endomycorrhizal spores alone in non-sterilized soil increased the survival percentage to 90% and gave the highest mean number and length of both shoots and roots, as well as leaves number. Moreover, they recorded the highest values of total chlorophyll, NPK and relative water contents. Key words: Stevia, micropropagation, mycorrhizae, mycorrhization helper bacteria.

Cultivable bacteria associated with infective propagules of arbuscular mycorrhizal fungi. Implications for mycorrhizal activity

This study aimed to isolate and characterize bacteria associated with surface-sterilized germinated propagules of arbuscular mycorrhizal (AM) fungi. It also aimed to evaluate their activity as mycorrhization helper bacteria (MHB) on the AM fungus Rhizophagus intraradices, which is commonly used in the formulation of bioinoculants. Most isolated bacteria did not significantly affect the viability and subsequent growth of mycelia. Azospirillum sp., Rhizobium etli, Bacillus megaterium, Bacillus sp., and Paenibacillus rhizosphaerae significantly enhanced pre-symbiotic variables (the re-growth/germination and the mycelia formed from AM propagules). P. rhizosphaerae, Azospirillum sp., and R. etli also increased extraradical mycelial length, mycorrhization percentages and the number of newly formed spores. The isolated MHB were characterized based on their starch-degrading ability, indole acetic acid production, phosphate solubilization, and inhibition of phytopathogenic fungal growth. Results suggest that some of the MHB studied, in association with viable AM propagules, could be potentially used as complex microbial inoculants for plant growth promotion.

Arthrinium phaeospermum isolated from Tuber borchii ascomata: the first evidence for a “Mycorrhization Helper Fungus”?

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.

Detection and quantification of a mycorrhization helper bacterium and a mycorrhizal fungus in plant-soil microcosms at different levels of complexity.

BackgroundHost plant roots, mycorrhizal mycelium and microbes are important and potentially interacting factors shaping the performance of mycorrhization helper bacteria (MHB). We investigated the impact of a soil microbial community on the interaction between the extraradical mycelium of the ectomycorrhizal fungus Piloderma croceum and the MHB Streptomyces sp. AcH 505 in both the presence and the absence of pedunculate oak microcuttings.ResultsSpecific primers were designed to target the internal transcribed spacer of the rDNA and an intergenic region between two protein encoding genes of P. croceum and the intergenic region between the gyrA and gyrB genes of AcH 505. These primers were used to perform real-time PCR with DNA extracted from soil samples. With a sensitivity of 10 genome copies and a linear range of 6 orders of magnitude, these real-time PCR assays enabled the quantification of purified DNA from P. croceum and AcH 505, respectively. In soil microcosms, the fungal PCR signal was not affected by AcH 505 in the absence of the host plant. However, the fungal signal became weaker in the presence of the plant. This decrease was only observed in microbial filtrate amended microcosms. In contrast, the PCR signal of AcH 505 increased in the presence of P. croceum. The increase was not significant in sterile microcosms that contained plant roots.ConclusionsReal-time quantitative PCR assays provide a method for directly detecting and quantifying MHB and mycorrhizal fungi in plant microcosms. Our study indicates that the presence of microorganisms and plant roots can both affect the nature of MHB-fungus interactions, and that mycorrhizal fungi may enhance MHB growth.

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.

The role of mycorrhization helper bacteria in the establishment and action of ectomycorrhizae associations.

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.

What are the mechanisms and specificity of mycorrhization helper bacteria?

What are the mechanisms and specificity of mycorrhization helper bacteria?

Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation

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.

Importance of mycorrhization helper bacteria cell density and metabolite localization for the Pinus sylvestris–Lactarius rufus symbiosis

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 (10(5), 10(7), 10(9) and 10(10) CFU mL(-1)), whereas, EJP67 only stimulated mycorrhiza formation within a narrow range of inoculum densities (10(7) and 10(9) 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 ( approximately 10 cells) within the mycorrhizal mantle, both at the root tip and along the length of the mycorrhizal short roots.

Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell. seedlings

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 Laccaria bicolor (S238), and seven Western Australian bacterial isolates from Laccaria fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaria 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 (Slf14) 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).

Suppression of common root pathogens by helper bacteria and ectomycorrhizal fungi in vitro

Root pathogens cause considerable loss of tree seedlings in nurseries and are generally difficult to control using conventional methods. Inoculation with ectomycorrhizal fungi may provide some suppression of pathogens. Bacteria (so-called mycorrhization helper bacteria) have been isolated that stimulate mycorrhiza formation on seedling roots and enhance seedling growth; however, their role in pathogen inhibition has not been explored. Four strains of helper bacteria were inoculated together with the ectomycorrhizal fungal species Laccaria bicolor, L. proxima and Suillus granulatus on culture plates to determine inhibition of the pathogens Fusarium oxysporum and Cylindrocarpon sp. Buffered medium was used to rule out acidification of the medium as a mechanism of inhibition. None of the ectomycorrhizal fungal species alone inhibited the growth of Fusarium but all showed slight inhibition of Cylindrocarpon growth. Helper bacterium strain MB3 (Bacillus subtilis) was effective in inhibiting both pathogens and, when inoculated with either L. proxima or S. granulatus, inhibition of Fusarium growth was enhanced over MB3 alone. With Cylindrocarpon, however, only S. granulatus inoculated along with MB3 showed enhanced inhibition over MB3 alone. The other three bacterial strains had little effect on the growth of Fusarium or Cylindrocarpon. More research is necessary to determine if these inhibitory effects are reproducible in situ.

Tansley Review No. 76 Helper bacteria: a new dimension to the mycorrhizal symbiosis.

The symbiotic establishment of mycorrhizal fungi on plant roots is affected in various ways by the other microorganisms of the rhizosphere, and more especially by bacteria. This review discusses the case of some of these bacteria which consistently promote mycorrhizal development, leading to the concept of 'mycorrhization' helper bacteria (MHBs). Examples of MHB evidence are given from the literature, with special reference to the Douglas fir (Pseudotsuga menzeisii Mirb. Franco) Laccaria laccata Scop, ex Fr. ectomycorrhizal combination which has been more extensively studied. The fungal specificity of some MHBs and the various mechanisms underlying their effect are discussed, considering five hypotheses: effects on the receptivity of the root, effects on the root-fungus recognition, effects on the fungal growth, modification of the rhizospheric soil, and effects on the germination of the fungal propagule. MHBs are then considered for their ecological and evolutionary implications, and examples of practical applications in forest nurseries are given: when added to the fungal inoculum, MHBs can improve the success of ectomycorrhizal inoculation of planting stocks with fungi selected for their outstanding growth stimulation after outplanting. The conclusion points out a number of fundamental questions which remain unanswered about mycorrhization helper bacteria and suggests some investigation priorities in this new field of mycorrhiza research. Contents Summary 197 I. Introduction 197 II. Evidence for helper bacteria 198 III. Fungus-specificity of MHBs 200 IV. Mechanisms underlying the MHB effect 201 V. Ecological and evolutionary implications of MHBs 205 VI. Practical applications of MHBs 206 VII. Conclusions and perspectives 208 Acknowledgements 208 References 208.

Les bactéries auxiliaires de la mycorhization: une nouvelle dimension de la symbiose mycorhizienne

The relatively recent evidences leading to the MHB concept (mycorrhization helper bacteria, which specificaly enhance mycorrhiza formation) ask many new questions about their degree of interdepende...

The fungus-specificity of mycorrhization helper bacteria (MHBs) used as an alternative to soil fumigation for ectomycorrhizal inoculation of bare-root Douglas-fir planting stocks with Laccaria laccata

Mycorrhization helper bacteria (MHBs) isolated and selected from the Douglas fir-Laccaria laccata symbiotic system have previously been shown to be fungus-specific: they promote ectomycorrhizal establishment of Laccaria laccata but inhibit mycorrhiza formation by other fungi. In this paper, two experiments in a nursery producing two years-old bare-root Douglas-fir planting stocks confirm the specificity of MHBs under field conditions. They also show that, by selectively helping the introduced L. laccata against the resident symbionts, MHBs are an interesting alternative (safer and easier) to soil fumigation for the success of routine controlled mycorrhization of planting stocks in forest nurseries.

Effects of substrate sterilization, fungicide treatment, and mycorrhization helper bacteria on ectomycorrhizal formation of pedunculate oak (Quercus robur) inoculated with Laccaria laccata in two peat bare-root nurseries

Pedunculate oak seedlings (Quercus robur) inoculated with the ectomycorrhizal fungus Laccaria lacata were grown for 1 year on fertilized sphagnum peat in two nurseries. Three factors affecting microbial populations in the substrate were studied, fungicide treatment of the seeds, peat disinfection before sowing (methyl bromide or steam pasteurization), and inoculation with mycorrhization helper bacteria. Treatment of acorns with Iprodione had no depressive effect on mycorrhiza formation. Both disinfection techniques were equivalent, stimulating or depressing mycorrhiza formation depending on the initial microflora in the peat. The introduction of two previously selected mycorrhization helper bacteria (one Pseudomonas fluorescens and one unidentified fluorescent pseudomonad), isolated from L. laccata sporocarps associated with Douglas fir—L. laccata ectomycorrhizas in other nurseries, significantly increased the mycorrhizal rate from 30 to 53% of the short roots. The implications of these results for the controlled mycorrhization of planting stocks and the specificity of mycorrhization helper bacteria are discussed.

Effect of dual inoculation of Douglas fir with the ectomycorrhizal fungus Laccaria laccata and mycorrhization helper bacteria (MHB) in two bare-root forest nurseries

Douglas fir (Pseudotsuga menziesii) seedlings in two bare-root forest nurseries were inoculated with the ectomycorrhizal fungus Laccaria laccata, together or not with one of five mycorrhization helper bacteria isolated from L. laccata sporocarps or mycorrhizas and previously selected by in vitro and glasshouse screenings. With the most efficient MHB isolates, when compared to the control with no bacteria, the percent of mycorrhizal short roots was increased from 60 to 90 or from 80 to 100, depending on the nursery, with inoculation doses as low as 106 living cells per m2. A dual inoculum made of calcium alginate beads containing the two microorganisms appears to be a valuable technique for increasing the efficiency of ectomycorrhizal inoculation of planting stocks in forest nurseries.

Mycorrhization helper bacteria associated with the Douglas fir-Laccaria laccata symbiosis: effects in aseptic and in glasshouse conditions

Summary — A range of bacterial isolates from Laccaria laccata mycorrhizas and sporocarps were tested for their effect on ectomycorrhizal development of Douglas fir with L laccata. The experiments were carried out in aseptic conditions and in the glasshouse under summer and winter conditions. Fourteen isolates increased mycorrhizal development after 16 wk of growth in the summer experiment. Seven bacterial isolates displayed a significant stimulating effect in the winter experiment. All bacterial isolates tested under aseptic conditions displayed a significant stimulating effect. In the winter experiment, the treatments without L laccata inoculation were contaminated by Thelephora terrestris (ectomycorrhizal basidiomycete), a natural contaminant in the glasshouse. Six bacterial isolates displayed a significant inhibiting effect towards ectomycorrhizal infection by T terrestris. Three isolates enhanced the ectomycorrhizal development of Douglas fir with L laccata in all experiments. It is confirmed that the inoculation techniques in forest nurseries could be improved by such mycorrhization helper bacteria (MHB). The results with T terrestris suggest that the mechanisms involved in interactions between bacteria and mycorrhizal establishment are partly fungus-specific. The results of the experiments in aseptic conditons suggest that the MHB act directly on the plant or/and on the fungus. Their stimulating effect is not the result of the suppression of root pathogens or other inhibitors of mycorrhizal infection. MHB could be used both for enhancing the infection by an introduced fungus and for reducing unwanted infection by inefficient symbionts such as T terrestris. Thus, the need for soil disinfection before inoculating might be reduced.