High potential of symbiotic interactions between native mycorrhizal fungi and the exotic tree Eucalyptus camaldulensis for phytostabilization of metal-contaminated arid soils.

Waste dumps generated by mining activities contain heavy metals that are dispersed into areas leading to significant environmental contamination. The objectives of this study were (i) to survey native plants and their associated AM fungal communities from waste soils in a Moroccan mine site and (ii) to follow Eucalyptus growth in soil collected from the waste-mine. AM spores from native plant species were collected from the mining site and the surrounding uncontaminated areas were multiplied and inoculated onto Eucalyptus camaldulensis. The results showed that (i) the native plant species recorded in the waste did not show an active metal uptake, (ii) the selected native plant species are associated with AM mycorrhizal fungi and (iii) the use of AM fungi adapted to these drastic conditions can improve the growth of the fast-growing tree, E. camaldulensis and its tolerance to high soil Cu content. In conclusion, it is suggested that in order to define efficient low-cost phytostabilization processes, the use of native resources (i.e., mixtures of native mycorrhizal fungi) in combination with fast-growing tree species such as Eucalyptus, could be used to optimize the establishment of a permanent cover plant in contaminated areas.

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.

The exotic legume tree species Acacia holosericea alters microbial soil functionalities and the structure of the arbuscular mycorrhizal community.

The response of microbial functional diversity as well as its resistance to stress or disturbances caused by the introduction of an exotic tree species, Acacia holosericea, ectomycorrhized or not with Pisolithus albus, was examined. The results show that this ectomycorrhizal fungus promotes drastically the growth of this fast-growing tree species in field conditions after 7 years of plantation. Compared to the crop soil surrounding the A. holosericea plantation, this exotic tree species, associated or not with the ectomycorrhizal symbiont, induced strong modifications in soil microbial functionalities (assessed by measuring the patterns of in situ catabolic potential of microbial communities) and reduced soil resistance in response to increasing stress or disturbance (salinity, temperature, and freeze-thaw and wet-dry cycles). In addition, A. holosericea strongly modified the structure of arbuscular mycorrhizal fungus communities. These results show clearly that exotic plants may be responsible for important changes in soil microbiota affecting the structure and functions of microbial communities.

Successful nodulation of Casuarina by Frankia in axenic conditions.

AIMS: In order to depict the fine interactions that lead to nodulation, absolute microbiological control of the symbiotic partners is required, i.e. the ability to obtain in vitro axenic nodulation, a condition that has never been fulfilled with the Casuarina-Frankia symbiosis. The effects of culture conditions on plant growth and nodule formation by Casuarina cunninghamiana were investigated. METHODS AND RESULTS: Axenic (capped tubes with different substrates), and nonaxenic cultures (Gibson tubes, pot cultures) were tested. In axenic conditions, C. cunninghamiana, inoculated with Frankia, had poor growth and did not form nodules at 6 weeks. Plants cultivated in Gibson tubes reached the four axillary shoots stage within 6 weeks and formed nodules 4 weeks after inoculation. Sand-pot cultures allowed us to relate the plant development stage at inoculation with nodulation. CONCLUSIONS: The sterile replacement of the cap by a plastic bag increased plant growth and enabled nodule formation 6 weeks after inoculation. The new system of plant culture allows the axenic nodule formation 6 weeks after inoculation. Nodulation behaviour is related to plant development and confinement. SIGNIFICANCE AND IMPACT OF THE STUDY: This axenic plant nodulation system is of major interest in analysing the roles of Frankia genes in nodulation pathways.

Improvement of Cupressus atlantica Gaussen growth by inoculation with native arbuscular mycorrhizal fungi.

AIMS: The study aimed to determine whether inoculation with native arbuscular mycorrhizal (AM) fungi could improve survival and growth of seedlings in degraded soils of Morocco. METHODS AND RESULTS: Soil samples were collected from the rhizosphere of Cupressus atlantica trees in the N'Fis valley (Haut Atlas, Morocco). AM spores were extracted from the soil, identified and this mixture of native AM fungi was propagated on maize for 12 weeks on a sterilized soil to enrich the fungal inoculum. Then C. atlantica seedlings were inoculated with and without (control) mycorrhizal maize roots, cultured in glasshouse conditions and further, transplanted into the field. The experiment was a randomized block design with one factor and three replication blocks. The results showed that a high AM fungal diversity was associated with C. atlantica; native AM fungi inoculation was very effective on the growth of C. atlantica seedlings in glasshouse conditions and this plant growth stimulation was maintained for 1 year after outplanting. CONCLUSIONS: Inoculation of C. atlantica with AM fungi increased growth and survival in greenhouse and field. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that use of native species of AM fungi may accelerate reforestation of degraded soils. Further studies have to be performed to determine the persistence of these mycorrhizae for a longer period of plantation and to measure the effects of this microbial inoculation on soil biofunctioning.

Responses of soil microbial catabolic diversity to arbuscular mycorrhizal inoculation and soil disinfection.

Although it is usually admitted that arbuscular mycorrhizal (AM) fungi are key components in soil bio-functioning, little is known on the response of microbial functional diversity to AM inoculation. The aims of the present study were to determine the influence of Glomus intraradices inoculum densities on plant growth and soil microflora functional diversity in autoclaved soil or non-disinfected soil. Microbial diversity of soil treatments was assessed by measuring the patterns of in situ catabolic potential of microbial communities. The soil disinfection increased sorghum growth, but lowered catabolic evenness (4.8) compared to that recorded in the non-disinfected soil (6.5). G. intraradices inoculation induced a higher plant growth in the autoclaved soil than in the non-disinfected soil. This AM effect was positively related to inoculum density. Catabolic evenness and richness were positively correlated with the number of inoculated AM propagules in the autoclaved soil, but negatively correlated in the non-disinfected soil. In addition, after soil disinfection and AM inoculation, these microbial functionality indicators had higher values than in the autoclaved or in the non-disinfected soil without AM inoculation. These results are discussed in relation to the ecological influence of AM inoculation, with selected fungal strains and their associated microflora on native soil microbial activity.

The ectomycorrhizal fungus Scleroderma bermudense alleviates salt stress in seagrape (Coccoloba uvifera L.) seedlings.

The purpose of this study was to test the capacity of the ectomycorrhizal (ECM) fungus, Scleroderma bermudense, to alleviate saline stress in seagrape (Coccoloba uvifera L.) seedlings. Plants were grown over a range (0, 200, 350 and 500 mM) of NaCl levels for 12 weeks, after 4 weeks of non-saline pre-treatment under greenhouse conditions. Growth and mineral nutrition of the seagrape seedlings were stimulated by S. bermudense regardless of salt stress. Although ECM colonization was reduced with increasing NaCl levels, ECM dependency of seagrape seedlings increased. Tissues of ECM plants had significantly increased concentrations of P and K but lower Na and Cl concentrations than those of non-ECM plants. Higher K concentrations in the leaves of ECM plants suggested a higher osmoregulating capacity of these plants. Moreover, the water status of ECM plants was improved despite their higher evaporative leaf surface. The results suggest that the reduction in Na and Cl uptake together with a concomitant increase in P and K absorption and a higher water status in ECM plants may be important salt-alleviating mechanisms for seagrape seedlings growing in saline soils.

Fluorescent pseudomonads occurring in Macrotermes subhyalinus mound structures decrease Cd toxicity and improve its accumulation in sorghum plants.

Cd-tolerant bacterial strains of fluorescent pseudomonads, mostly belonging to Pseudomonas monteillii, were isolated from termite mound soil (Macrotermes subhyalinus, a litter-forager and fungus-growing termite), in a Sudanese shrubby savanna, Burkina Faso. Such large mounds appeared as sites of great bacterial diversity and could be considered as hot spots of metal-tolerant fluorescent pseudomonads. Microbial isolates were inoculated to Sorghum plants (S. bicolor) in glasshouse experiments with soil amended with CdCl(2) (560 mg Cd kg(-1) soil). Microbial functional diversity was assessed at the end of the experiment by measurement of in situ patterns of catabolic potentials. All the bacteria isolates significantly improved the shoot and total biomass of sorghum plants compared to the control. Results concerning root biomass were not significant with some strains. Arbuscular mycorrhiza (AM) was greatly reduced by CdCl(2) amendment, and fluorescent pseudomonad inoculation significantly increased AM colonisation in the contaminated soil. The bacterial inoculation significantly improved Cd uptake by sorghum plants. Measurement of catabolic potentials on 16 substrates showed that the microbial communities were different according to the soil amendment. Soils samples inoculated with pseudomonad strains presented a higher use of ketoglutaric and hydroxybutiric acids, as opposed to fumaric acid in soil samples not inoculated. It is suggested that fluorescent pseudomonads could act indirectly in such metabolic processes by involving a lower rate of degradation of citric acid, in line with the effect of small organic acid on phytoextraction of heavy metals from soil. This is a first contribution to bioremediation of metal-contaminated sites with soil-to-plant transfer, using termite built structures. Further data are required on the efficiency of the bacterial strains isolated and on the processes involved.

Contrasting responses to ectomycorrhizal inoculation in seedlings of six tropical African tree species.

Five caesalpinioid legumes, Afzelia africana, Afzelia bella, Anthonotha macrophylla, Cryptosepalum tetraphylum and Paramacrolobium coeruleum, and one Euphorbiaceae species, Uapaca somon, with a considerable range in seed sizes, exhibited different responses to inoculation by four species of ectomycorrhizal (ECM) fungi, Scleroderma dictyosporum, S. verrucosum, Pisolithus sp. and one thelephoroid sp. in greenhouse conditions. Thelephoroid sp. efficiently colonized seedlings of all of the five caesalpinioid legumes except U. somon, but provided no more growth benefit than the other fungi. Thelephoroid sp. and S. dictyosporum colonized seedlings of U. somon poorly, but stimulated plant growth more than the other fungi. The relative mycorrhizal dependency (RMD) values of the caesalpinioid legumes were never higher than 50%, whilst U. somon had RMD values ranging from 84.6 to 88.6%, irrespective of the fungal species. The RMD values were negatively related to seed mass for all plant species. Potassium concentrations in leaves were more closely related than phosphorus to the stimulation of seedling biomass production by the ECM fungi. Our data support the hypothesis that African caesalpinioid legumes and euphorbe tree species with smaller seeds show higher RMD values than those with the larger seeds.

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

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.

Relative importance of the endomycorrhizal and (or) ectomycorrhizal associations in Allocasuarina and Casuarina genera.

This work was carried out to determine the relative importance of the endomycorrhizal and (or) ectomycorrhizal association in species of Casuarina and Allocasuarina. Under axenic conditions, Pisolithus and Scleroderma isolates formed ectomycorrhizas with a mantle and a Hartig net on Allocasuarina verticillata but failed to form a Hartig net on Casuarina glauca. In a controlled soil system, C. glauca was inoculated with the endomycorrhizal fungus Glomus intraradices Schenck & Smith, and A. verticillata was inoculated with Pisolithus albus IR100 Bougher & Smith and (or) G. intraradices. Both symbionts significantly stimulated growth in both plant species. For A. verticillata, its growth response to ectomycorrhizal inoculation was higher than to endomycorrhizal inoculation. When both symbionts were inoculated, antagonism among the fungal isolates was observed with a higher ectomycorrhizal colonization. These results showed that A. verticillata was ectomycorrhizal dependent, whereas C. glauca was endomycorrhizal dependent. From a practical point of view, this study shows the importance of selecting compatible mycorrhizal fungi for developing successful inoculation programmes. In addition, it would help to further research and determine the effect of ecto- and endo-mycorrhizal symbiosis on the formation and function of N2-fixing actinorhizal nodules.

A mycorrhiza helper bacterium enhances ectomycorrhizal and endomycorrhizal symbiosis of Australian Acacia species.

The aims of this study were to test the effects of a mycorrhiza helper bacterium (MHB), Pseudomonas monteilii strain HR13 on the mycorrhization of (1) an Australian Acacia, A. holosericea, by several ectomycorrhizal fungi or one endomycorrhizal fungus Glomus intraradices, and (2) several Australian Acacia species by Pisolithus alba strain IR100 under glasshouse conditions. Bacterial inoculant HR13 significantly promoted ectomycorrhizal colonization for all the Acacia species, from 45.8% ( A. mangium) to 70.3% ( A. auriculiformis). A stimulating effect of HR13 on the ectomycorrhizal establishment was recorded with all the fungal isolates (strains of Pisolithus and Scleroderma). The same effect of bacteria on the frequency of endomycorrhizal colonization of A. holosericea seedlings by G. intraradices with vesicles and hyphae frequencies was recorded. The stimulation of saprophytic fungal growth by MHB is usually the main mechanism that could explain this bacterial effect on mycorrhizal establishment. MHB could stimulate the production of phenolic compounds such as hypaphorine and increase the aggressiveness of the fungal symbiont. However, no significant effect of MHB on fungal growth was recorded with Scleroderma isolates under axenic conditions but positive bacterial effects were observed with Pisolithus strains. From a practical viewpoint, it appears that MHB could stimulate the mycorrhizal colonization of Australian Acacia species with ectomycorrhizal or endomycorrhizal fungi, and could also facilitate controlled mycorrhization in nursery practices where Acacia species are grown for forestation purposes.

Arbuscular mycorrhizal symbiosis changes the colonization pattern of Acacia tortilis spp. Raddiana rhizosphere by two strains of rhizobia.

The aim of the study was to assess the effect of the mycorrhizosphere of A. tortillis spp. raddiana mycorrhized with Glomus intraradices on the root nodulation by Sinorhizobium terangae (ORS 1009) and/or Mesorhizobium plurifarium (ORS 1096) in two different culture substrates (sandy soil and sand). The endomycorrhizal fungus only stimulated plant growth in the sandy soil. Moreover, arbuscular mycorrhizal infection enhanced the nodulation process in both culture substrates. Beside the stimulatory effects of the mycorrhizosphere on both rhizobia development, fungal symbiosis induces two different dynamics of each bacterial strains in the sand-grown plants. These results suggest specific relationships could occur during the development of the tripartite symbiosis, at physiological and molecular level. From a practical point of view, the role of arbuscular mycorrhizas in improving nodulation and N2 fixation is universally recognized. The fungal symbiosis could modify the development of bacterial inoculants along the root systems. This effect is of particular interest in the controlled inoculation of selected rhizobia.

Cuticle heterogeneity as exhibited by Pasteuria spore attachment is not linked to the phylogeny of parthenogenetic root-knot nematodes (Meloidogyne spp.).

The cuticle is a major barrier prohibiting the infection of nematodes against micro-organisms. The attachment of bacterial spores of the nematode hyperparasite Pasteuria penetrans (PP1) to field populations of root-knot nematodes (RKN, Meloidogyne spp.) from Burkino Faso, Ecuador, Greece, Malawi, Senegal and Trinidad and Tobago were assayed in standard attachment tests. The attachment of spore population PP1 to different field populations of root-knot nematode showed that the rates of attachment differed between countries. Similar tests were also undertaken on P. penetrans spores from these countries against 2 species of RKN, M. incognita and M. arenaria. The results showed a high degree of variability in spore attachment with no clear distinction between the 2 species of nematode. It has been hypothesized that Pasteuria spore attachment is linked to nematode species designations and this study clearly shows that this is not the case. Further tests showed that variation in spore attachment was not linked to nematode phylogeny. The results therefore beg the question of how do parthenogenetic root-knot nematodes maintain cuticle variability in the face of such an aggressive hyperparasite.