Growth dynamic of dual culture systems comprising ectomycorrhizal fungi and mycorrhiza helper bacteria

Ectomycorrhizal fungi are ubiquitous root symbionts that associate with the majority of forest trees and often have a crucial role on plant survival and growth in impoverished soils. Within the vast community of soil microorganisms, the mycorrhiza helper bacteria (MHB) are recognizably one of the groups that most directly affect fungal growth and mycorrhiza establishment. Although their positive effect on the mycorrhizal partnership has been previously reported, the specificity of such association is high and the dynamics not yet fully understood. Many studies evaluated the impact of bacteria on fungal growth but the reciprocal has received little attention. The present study aimed to evaluate the growth dynamics of co-culturing fungi and bacteria, under both perspectives. Bacillus pumilus was chosen for its capability to promote the growth of Suillus granulatus in a dual culture system with a low nutrient medium. To assess the impact of fungal exudates on bacterial growth, different quantities of fungal culture medium were added to MMN medium containing a Bacillus pumilus inoculum and bacterial growth was monitored. To evaluate the influence of Bacillus pumilus on fungal growth, an experiment was setup where the bacterium was added at two different phases of fungal growth, lag phase and exponential phase. A control without bacteria was also performed. Bacterial growth was monitored through OD readings and fungal growth was assessed through dry weight and ergosterol content. The experience was held for 33 days, with sampling every three days. Triplicates for each treatment were performed. Results from the first experiment showed that fungal exudates influenced the growth of bacteria with a pronounced extension of the lag phase. In the second experiment, fungal growth was completely inhibited when bacteria was added at the fungal lag phase. A different outcome was observed when the bacterium was added in the exponential phase. Results suggest that the regulation of the concentration at which the bacterium is present may be a key factor to optimization of the use of bacteria as MHB

Bioremediation on anthropogenic affected areas: Ectomycorrhizal and plant growth bacteria as promoters of pine establishment

The recovery of damaged areas due to inadequate farming policies and increased industrial sediment deposition, have contaminated not only soil and surrounding areas but also other natural resources. The potential use of disturbed sites for agriculture and forestry is jeopardised and their remediation is critical and expensive. The utilization of biotechnological tools, such as plant growth promoting bacteria (PGPB) and ectomycorrhizal fungi (ECM) could help remediation of such soils as they can be used as plant facilitators for land recovery. The aim of this study was to assess the potential of PGPB and ECM to enhance the growth of Pinus pinaster in antropogenic sediments and forest soil. Pine seedlings were inoculated with Suillus bovinus, Pisolithus tinctorius and Paxillus involutus, and co-inocualted with Bacillus spp. and Mesorhizobium spp. Plants were harvested after 6 month growth and parametric and nutritional data determined. Results show that P. involutus increased seedling growth(height) in industrial sediments soil, whereas in forest soil, plant performance was higher with S. bovinus. The effect of inoculation on the fungal communityin seedling roots and bacterial rhizosphere was also analysed by PCR-DGGE and differences arose between inoculated and uninoculated soil, indicating that PGPB and ECM may significantly influence the plant growth performance over a period of time. The study shows that PGPB and ECM fungi may be used as a biotechnology tool contributing to the successful plant establishment in disturbed environments

Selection of high performance strains of edible mycorrhizal fungi for improved abiotic stress resistance

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Reforestation of burned stands: the effect of ectomycorrhizal fungi on Pinus pinaster establishment

The area occupied by Pinus pinaster in Portugal is rapidly diminishing because of forest fires. Ectomycorrhizal fungi form obligate, mutually beneficial associations with P. pinaster which improve plant growth and resistance to adverse conditions. The aim of this work was to assess whether native ectomycorrhizal fungi could be a useful tool in the reforestation of burned areas. The work was conducted in a forest nursery greenhouse, where P. pinaster seedlings were inoculated with compatible ectomycorrhizal fungal isolates: Suillus bovinus, Pisolithus tinctorius, Rhizopogon roseolus, and a mixture of the three fungi, using burned and unburned forest soil as substrate. Inoculation significantly enhanced the growth of P. pinaster, with R. roseolus proving to be the most effective in burned soil, with an 8-fold increase in plant fresh weight. Overall, inoculation stimulated growth most in burned than in unburned soil. This study suggests that inoculation with selected ectomycorrhizal fungi in containerised nurseries can be an advantageous approach for the successful establishment of P. pinaster in burned soil. The obtained results point out to the interest of extending these studies into fire-impacted areas, using ectomycorrhizal fungi as a biological tool.info:eu-repo/semantics/acceptedVersio

Succession dynamics of ectomycorrhizal fungi in inoculated Quercus rubra seedlings – a field study

Selected ectomycorrhizal (ECM) fungi may have a beneficial effect on the establishment of nursery grown seedlings and may influence their survival on the first years. By increasing seedling capacity to capture nutrients, ECM fungi often contribute to a successful plant establishment. We investigated the performance of pre-inoculated Quercus rubra seedlings on a reforestation site and monitored ECM fungal persistence and succession dynamics between selected ECM fungi and native fungal community. Nursery grown Q. rubra seedlings inoculated with a mixture of ECM fungi (Cenococcum geophilum, Hebeloma crustuliniforme, H. mesophaeum, H. velutipes, Paxillus involutus, Scleroderma citrinum) and non-inoculated control oak seedlings, were transplanted to Serra da Cabreira in Northern Portugal. Two years after planting, the subsistence of the inoculated ECM fungi was assessed using ITS-DGGE. Results demonstrated that inoculation with selected ECM fungi at nursery stage promoted the establishment and improved the growth performance of out planted oak seedlings. C. geophilum was significantly associated with inoculated saplings but other ECM fungi species were also found. Moreover, ECM fungal communities of inoculated and non-inoculated samples are significantly different. Further monitoring is required to increase knowledge on the persistence of ECM fungal communities and their succession dynamics

Two genotypes of mycorrhizal Pinus pinaster respond differently to cadmium contamination

Fertilization is one of the main anthropogenic sources of Cd accumulation in agricultural soils and when toxic levels have been reached, food crop production is no longer viable. Adequate strategies for the forestation of agricultural metal contaminated sites are of vital importance. The aim of this work was to evaluate the response of two different genotypes of P. pinaster (A and B) to Cd contamination and to assess how inoculation with ectomycorrhizal fungi influenced each genotype. Seedlings were exposed to soil contaminated at different levels of Cd. At 30 mg Cd kg-1 non-inoculated genotype A accumulated more Cd in the shoots. At the lowest Cd concentration S. bovinus decreased Cd shoot concentration and increased aboveground development in both genotypes. At the highest Cd dosage inoculation with R. roseolus decreased Cd concentration in the roots of genotype B whereas the opposite occurred in genotype A. The results from this study suggest that the selection of an adequate combination between genotype and associated mycobionts may be an important biotechnological tool to enhance the efficiency of forestation and phytoremediation processes of degraded land using P. pinaster

Inoculation with ectomycorrhizal fungi affects Pinus pinaster performance under cadmium exposure

Afforestation of contaminated sites can represent a valuable approach to restore degraded ecosystems. Studies on the response of woody species to heavy metal contamination in soil are scarce compared to crop species. Cadmium is one of the most toxic heavy metals and its hazardous effects are well known. The aim of this work was to evaluate Pinus pinaster performance on Cd contaminated soil (15 and 30 mg Cd kg-1) and determine whether inoculation with two ectomycorrhizal fungi, Suillus bovinus and Rhizopogon roseolus influenced such response. Regarding non-inoculated seedlings, Cd exposure led to a lower shoot biomass and metal accumulation on the root system was proportional to its concentration in the soil. Inoculation with S. bovinus was the most favorable treatment for P. pinaster development by enhancing shoot development up to 1.3-fold in contaminated soil. Inoculation with R. roseolus increased Cd concentration in the shoots with no significant effect in any of the biometric traits studied. Metal accumulation on the shoots and roots of P. pinaster seedlings was significantly affected by the interaction between mycorrhizal inoculation and the Cd concentration to which the seedlings were exposed. Results from this study show that inoculation with selected ECM fungi can influence the performance of P. pinaster under Cd exposure and that this biotechnological tool could be of great value for plant establishment in contaminated sites

The interaction between ectomycorrhizal fungi and growth promoting bacteria in plant development from in-vitro to the field

There is currently an array of biotechnological tools which may positively influence plant development and establishment. Symbiosis with ectomycorrhizal fungi is known to improve plant health by increasing nutrient and water uptake and alleviating environmental stresses. Some rhizosphere bacteria are also proven to act as plant stimulating agents, health managers and growth promoters. The use of a combined inoculum using both microorganisms has great potential in forestry. Nevertheless, the interaction between fungi and bacteria is highly specific and bacteria are known not only to enhance fungal growth (mycorrhiza promoting bacteria), but also to strongly inhibit it. It is therefore important to evaluate the compatibility between ectomycorrhizal fungi and bacteria and to determine the impact of such combination in plant development when aiming at the use of optimized microbial inoculum for enhancing plant growth. In the present work we investigated the use of a dual inoculation system, consisting of a bacterial strain from the genus Mesorhizobium in combination with ectomycorrhizal fungi, from in-vitro tests to a nursery stage followed by transplantation to the field. Approximately 5000 seedlings were used in these experiments comprising Quercus suber, Quercus robur, Quercus rubra, Pinus pinaster and Pinus pinea. Seedlings were grown for 9 months in a commercial forest greenhouse and then transplanted to 4 locations in Portugal. The first sampling was made before field transplantation. Results showed that the combined use of fungi-bacteria inoculum significantly influenced plant growth and its effect was different from that of each individual microbial partner, emphasizing the strong interaction between microorganisms