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.     

Arbuscular mycorrhizal symbiosis can counterbalance the negative influence of the exotic tree species Eucalyptus camaldulensis on the structure and functioning of soil microbial communities in a sahelian soil

The hypothesis of the present study was that bacterial communities would differentiate under Eucalyptus camaldulensis and that an enhancement of arbuscular mycorrhizal (AM) density would minimize this exotic plant species effect. Treatments consisted of control plants, preplanting fertilizer application and AM inoculation. After 4 months of culture in autoclaved soil, E. camaldulensis seedlings were either harvested for growth measurement or transferred into containers filled with the same soil but not sterilized. Other containers were kept without E. camaldulensis seedlings. After 12 months, effects of fertilizer amendment and AM inoculation were measured on the growth of Eucalyptus seedlings and on soil microbial communities. The results clearly show that this plant species significantly modified the soil bacterial community. Both community structure (assessed by denaturing gradient gel electrophoresis profiles) and function (assessed by substrate-induced respiration responses including soil catabolic evenness) were significantly affected. Such changes in the bacterial structure and function were accompanied by disturbances in the composition of the herbaceous plant species layer. These results highlight the role of AM symbiosis in the processes involved in soil bio-functioning and plant coexistence and in afforestation programmes with exotic tree species that target preservation of native plant diversity.     

Soil Bacterial Diversity Responses to Root Colonization by an Ectomycorrhizal Fungus are not Root-Growth-Dependent

The hypothesis tested in this present study was that the ectomycorrhizosphere effect on the bacterial community was not root-growth-dependent. The impacts of ectomycorrhizal infection (Pisolithus albus COI007) and a chemical fertilization to reproduce the fungal effect on root growth were examined on (1) the structure of bacterial community and (2) fluorescent pseudomonad and actinomycete populations in the mycorrhizosphere of Acacia auriculiformis using both culture-independent and culture-dependent methods. A. auriculiformis plants were grown in disinfested soil in pots with or without addition of the ectomycorrhizal fungus or N/P/K fertilization (to reproduce the fungal effect on root growth) for 4 months and then transferred to 20-L pots filled with nondisinfested sandy soil. The fungal and fertilizer applications significantly improved the plant growth after 4-month culture in the disinfested soil. In the nondisinfested cultural substrate, these positive effects on plant growth were maintained. The total soil microbiota was significantly different within the treatments as revealed from DNA analysis [denaturing gradient gel electrophoresis (DGGE)]. The structure of fluorescent pseudomonad populations was also affected by fungal and fertilizer applications. In contrast, no qualitative effect was observed for the actinomycete communities within each treatment, but fungal inoculation significantly decreased the number of actinomycetes compared to the fertilizer application treatment. These results show that the mycorrhizosphere effect is not root-growth-dependent but is mainly due to the presence of the ectomycorrhizal fungus and more particularly to the extramatrical mycelium.     

Improvement of cowpea productivity by rhizobial and mycorrhizal inoculation in Burkina Faso

Cowpea is one of the most important food legume crops in Burkina Faso. It is able to associate with arbuscular mycorrhizal fungi (AMF) and rhizobia. This dual symbiosis improves nitrogen and phosphorus nutrient uptake in cowpea. As the application of exotic inoculants frequently lacks positive responses in field experiments, this study set out to select well-adapted native symbiotic rhizobial and AMF strains. Soil samples were collected from six study sites in three different climatic zones of Burkina Faso to investigate their native symbiotic strains. Soil-extraction of native spores led to the identification of four AMF genera (Scutellospora, Gigaspora, Glomus and Entrophospora) by morpho-anatomical characterization. The two most effective cowpea fungal strains were selected after spore isolation from field-collected soils, multiplication on maize roots and inoculation on cowpea seedlings in a greenhouse experiment. Cowpea-nodulating rhizobial strains were trapped in the greenhouse by planting cowpea seeds in collected soil samples and the strains were characterized using molecular methods. This characterization led to the rhizobial isolates being classified in four clusters on the phylogenetic tree (using the Maximum-Likelihood Phylogenies method). All strains belonged to the Bradyrhizobium genus and most of them were included in the B. japonicum branch. Some groups were clearly distinct species already identified and may be new species. The two most effective strains for cowpea yield improvement in the field were selected after cowpea inoculation in a greenhouse experiment. The inoculation design in the field experiment consisted of four single inoculation treatments, either rhizobial or mycorrhizal, along with four dual inoculations, one treatment with chemical fertilizers, and one uninoculated control. The results showed that cowpea productivity was significatively improved by dual inoculation with native rhizobial and mycorrhizal strains, reaching the same level as the application of commonly used chemical fertilizers [Nitrogen, Phosphorus and Potassium fertilizers (NPK)]. In addition, dual inoculation resulted in the highest iron content in cowpea leaves.     

Responses of Pinus halepensis growth, soil microbial catabolic functions and phosphate-solubilizing bacteria after rock phosphate amendment and ectomycorrhizal inoculation

We examined the effects of an ectomycorrhizal (EM) fungus, Pisolithus sp., on of the growth of Pinus halepensis (Allepo pine) seedlings, soil microbial functions and rock phosphate solubilization in a un-disinfected soil amended or not with a Moroccan rock phosphate. Allepo pine seedlings were inoculated with an EM fungus (Pisolithus sp. strain PH4) isolated from a P. halepensis plantation and selected for its high ability to mobilize P from an inorganic form of phosphate. After 4 month’s culture in a disinfected substrate, plants were transferred in 10 l-containers filled with a natural forest soil and amended or not with rock phosphate powder. After 12 month’s culturing, the growth, needle nutrient concentrations of P. halepensis plants were measured. Soil microbial catabolic diversity was assessed by measuring CO₂ production of substrate induced respiration responses. Fluorescent pseudomonads were isolated from each soil treatment and tested in axenic conditions for their ability to solubilize a source of inorganic phosphate. The results clearly showed that (i) P. halepensis growth was greatly promoted by the presence of the ectomycorrhizal fungus Pisolithus strain PH4 in a disinfected soil/vermiculite mixture and in a non disinfected soil, (ii) ectomycorrhizal inoculation induced significant changes in the functions of soil microbial communities and selected microorganisms potentially beneficial to the plant growth (i.e. phosphate-solubilizing fluorescent pseudomonad) and (iii) rock phosphate solubilisation was mainly dependent on EM inoculation and mycorrhizosphere microorganisms. These results were in accordance with previous studies where it was demonstrated that EM symbiosis has a beneficial effect on plant growth through a direct effect on the host plant but also an indirect effect via a selective pressure on soil microbiota that favours microorganisms potentially beneficial to plant growth.     

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.     

Differing Courses of Genetic Evolution of Bradyrhizobium Inoculants as Revealed by Long-Term Molecular Tracing in Acacia mangium Plantations

Introducing nitrogen-fixing bacteria as an inoculum in association with legume crops is a common practice in agriculture. However, the question of the evolution of these introduced microorganisms remains crucial, both in terms of microbial ecology and agronomy. We explored this question by analyzing the genetic and symbiotic evolution of two Bradyrhizobium strains inoculated on Acacia mangium in Malaysia and Senegal 15 and 5 years, respectively, after their introduction. Based on typing of several loci, we showed that these two strains, although closely related and originally sampled in Australia, evolved differently. One strain was recovered in soil with the same five loci as the original isolate, whereas the symbiotic cluster of the other strain was detected with no trace of the three housekeeping genes of the original inoculum. Moreover, the nitrogen fixation efficiency was variable among these isolates (either recombinant or not), with significantly high, low, or similar efficiencies compared to the two original strains and no significant difference between recombinant and nonrecombinant isolates. These data suggested that 15 years after their introduction, nitrogen-fixing bacteria remain in the soil but that closely related inoculant strains may not evolve in the same way, either genetically or symbiotically. In a context of increasing agronomical use of microbial inoculants (for biological control, nitrogen fixation, or plant growth promotion), this result feeds the debate on the consequences associated with such practices.     

Litter-forager termite mounds enhance the ectomycorrhizal symbiosis between Acacia holosericea A. Cunn. Ex G. Don and Scleroderma dictyosporum isolates

The hypothesis of the present study was that the termite mounds of Macrotermes subhyalinus (MS) (a litter-forager termite) were inhabited by a specific microflora that could enhance with the ectomycorrhizal fungal development. We tested the effect of this feeding group mound material on (i) the ectomycorrhization symbiosis between Acacia holosericea (an Australian Acacia introduced in the sahelian areas) and two ectomycorrhizal fungal isolates of Scleroderma dictyosporum (IR408 and IR412) in greenhouse conditions, (ii) the functional diversity of soil microflora and (iii) the diversity of fluorescent pseudomonads. The results showed that the termite mound amendment significantly increased the ectomycorrhizal expansion. MS mound amendment and ectomycorrhizal inoculation induced strong modifications of the soil functional microbial diversity by promoting the multiplication of carboxylic acid catabolizing microorganisms. The phylogenetic analysis showed that fluorescent pseudomonads mostly belong to the Pseudomonads monteillii species. One of these, P. monteillii isolate KR9, increased the ectomycorrhizal development between S. dictyosporum IR412 and A. holosericea. The occurrence of MS termite mounds could be involved in the expansion of ectomycorrhizal symbiosis and could be implicated in nutrient flow and local diversity.     

Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two Bradyrhizobium strains and Acacia holosericea growth

Abstract Two strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions. Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium. After 4 months culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated. PCR/RFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia. When both Bradyrhizobium isolates were inoculated together, but without P. albus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, whereas Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment. Therefore Aust 13C had a high competitive ability and a great persistence in soil. The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp. root nodules. Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other. From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N₂ fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.