Ectomycorrhizal Fungal Inoculation Research Articles

Pinus massoniana somatic embryo maturation, mycorrhization of regenerated plantlets and its resistance to Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a major quarantine forest disease that poses a threat to various pine species, including Pinus massoniana (masson pine), worldwide. Breeding of PWN-resistant pine trees is an important approach to prevent the disease. To expedite the production of PWN-resistant P. massoniana accessions, we investigated the effects of maturation medium treatments on somatic embryo development, germination, survival, and rooting. Furthermore, we evaluated the mycorrhization and nematode resistance of regenerated plantlets. Abscisic acid was identified as the main factor affecting maturation, germination, and rooting of somatic embryos in P. massoniana, resulting in a maximum of 34.9 ± 9.4 somatic embryos per ml, 87.3 ± 9.1% germination rate, and 55.2 ± 29.3% rooting rate. Polyethylene glycol was identified as the main factor affecting the survival rate of somatic embryo plantlets, with a survival rate of up to 59.6 ± 6.8%, followed by abscisic acid. Ectomycorrhizal fungi inoculation with Pisolithus orientalis enhanced the shoot height of plantlets regenerated from embryogenic cell line (ECL) 20-1-7. Ectomycorrhizal fungi inoculation also improved the survival rate of plantlets during the acclimatization stage, with 85% of mycorrhized plantlets surviving four months after acclimatization in the greenhouse, compared with 37% non-mycorrhized plantlets. Following PWN inoculation, the wilting rate and the number of nematodes recovered from ECL 20-1-7 were lower than those recovered from ECL 20-1-4 and 20-1-16. The wilting ratios of mycorrhizal plantlets from all cell lines were significantly lower than those of non-mycorrhizal regenerated plantlets. This plantlet regeneration system and mycorrhization method could be used in the large-scale production of nematode-resistance plantlets and to study the interaction between nematode, pines, and mycorrhizal fungi.

The Relationship between Ectomycorrhizal Fungi, Nitrogen Deposition, and Pinus massoniana Seedling Nitrogen Transporter Gene Expression and Nitrogen Uptake Kinetics.

Analyzing the molecular and physiological processes that govern the uptake and transport of nitrogen (N) in plants is central to efforts to fully understand the optimization of plant N use and the changes in the N-use efficiency in relation to changes in atmospheric N deposition changes. Here, a field experiment was conducted using the ectomycorrhizal fungi (EMF), Pisolithus tinctorius (Pt) and Suillus grevillei (Sg). The effects of N deposition were investigated using concentrations of 0 kg·N·hm-2a-1 (N0), a normal N deposition of 30 kg·N·hm-2a-1 (N30), a moderate N deposition of 60 kg·N·hm-2a-1 (N60), and a severe N deposition of 90 kg·N·hm-2a-1 (N90), with the goal of examining how these factors impacted root activity, root absorbing area, NH4+ and NO3- uptake kinetics, and the expression of ammonium and nitrate transporter genes in Pinus massoniana seedlings under different levels of N deposition. These data revealed that EMF inoculation led to increased root dry weight, activity, and absorbing area. The NH4+ and NO3- uptake kinetics in seedlings conformed to the Michaelis-Menten equation, and uptake rates declined with increasing levels of N addition, with NH4+ uptake rates remaining higher than NO3- uptake rates for all tested concentrations. EMF inoculation was associated with higher Vmax values than were observed for non-mycorrhizal plants. Nitrogen addition resulted in the upregulation of genes in the AMT1 family and the downregulation of genes in the NRT family. EMF inoculation under the N60 and N90 treatment conditions resulted in the increased expression of each of both these gene families. NH4+ and NO3- uptake kinetics were also positively correlated with associated transporter gene expression in P. massoniana roots. Together, these data offer a theoretical foundation for EMF inoculation under conditions of increased N deposition associated with climate change in an effort to improve N absorption and transport rates through the regulation of key nitrogen transporter genes, thereby enhancing N utilization efficiency and promoting plant growth. Synopsis: EMF could enhance the efficiency of N utilization and promote the growth of Pinus massoniana under conditions of increased N deposition.

The Effects of Suillus luteus Inoculation on the Diversity of Fungal Communities and Their Structures in the Soil under Pinus massoniana Located in a Mining Area

As important decomposers and plant symbionts, soil fungal communities play a major role in remediating heavy-metal-polluted soils. However, the diversity and structures of fungal communities generally remain unclear in mining areas. This study aimed to assess the rhizospheric fungal-community composition of Masson pine (Pinus massoniana) in the lead-zinc mining area of Suxian District, Hunan Province, China. This experiment undertook the following three treatments: Masson pine inoculated with or not inoculated with Suillus luteus, and bulk soil without plants as a control. The results thereof showed that inoculation of ectomycorrhizal fungi could enlarge plants’ capability to absorb heavy metals and secrete soil enzymes. The richness and diversity of fungi in the rhizospheric soil were significantly higher than of those in the bulk soil (p < 0.05), but no significant difference was noted between the rhizospheric soils inoculated with and not inoculated with ectomycorrhizal (ECM) fungi as the community structure changed. The rhizospheric fungi belonged to 6 phyla, 25 classes, 65 orders, 115 families, and 150 genera, and the dominant phyla were Chytridiomycota (50.49%), Ascomycota (38.54%), and Basidiomycota (9.02%). Through use of LEfSe and heatmapping, the relative abundances of Suillus, Paraglomus, Agaricus, and Tulasnella were found to be the highest in the soil with ECM fungus inoculation. RDA showed that the community structure nearly changed with ECM-fungus inoculation; this was significantly related to soil water content, the carbon–nitrogen ratio, bulk density, available potassium, and soil enzymes. Altogether, inoculation with ECM fungi may change the habitation environments of microorganisms and dominant fungi in soil, providing keystone screenings in heavy-metal-contaminated mining areas.

Ectomycorrhizal and non-mycorrhizal rhizosphere fungi increase root-derived C input to soil and modify enzyme activities: A 14 C pulse labelling of Picea abies seedlings.

Consequences of interactions between ectomycorrhizal fungi (EcMF) and non-mycorrhizal rhizosphere fungi (NMRF) for plant carbon (C) allocation belowground and nutrient cycling in soil remain unknown. To address this topic, we performed a mesocosm study with Norway spruce seedlings [Picea abies (L.) H. Karst] inoculated with EcMF, NMRF, or a mixture of both (MIX). 14 CO2 pulse labelling of spruce was applied to trace and visualize the 14 C incorporation into roots, rhizohyphosphere and hyphosphere. Activities and localization of enzymes involved in the C, nitrogen (N) and phosphorus (P) cycling were visualized using zymography. Spruce seedlings inoculated with EcMF and NMRF allocated more C to soils (EcMF: 10.7%; NMRF: 3.5% of total recovered C) compared to uninoculated control seedlings. The 14 C activity in the hyphosphere was highest for EcMF and lowest for NMRF. In the presence of both, NMRF and EcMF (MIX), the 14 C activity was 64% lower compared with EcMF inoculation alone. This suggests a suppressed C allocation via EcMF likely due to the competition between EcMF and NMRF for N and P. Furthermore, we observed 57% and 49% higher chitinase and leucine-aminopeptidase activities in the rhizohyphosphere of EcMF compared to the uninoculated control, respectively. In contrast, β-glucosidase activity (14.3 nmol cm-2 h-1 ) was highest in NMRF likely because NMRF consumed rhizodeposits efficiently. This was further supported by that enzyme stoichiometry in soil with EcMF shifted to a higher investment of nutrient acquisition enzymes (e.g., chitinase, leucine-aminopeptidase, acid phosphatase) compared to NMRF inoculation, where investment in β-glucosidase increased. In conclusion, the alleviation of EcMF from C limitation promotes higher activities of enzymes involved in the N and P cycle to cover the nutrient demand of EcMF and host seedlings. In contrast, C limitation of NMRF probably led to a shift in investment towards higher activities of enzymes involved in the C cycle.

Photosynthetic product allocations of Pinus massoniana seedlings inoculated with ectomycorrhizal fungi along a nitrogen addition gradient.

Quantifying the allocation of photosynthetic products among different carbon (C) pools is critical for understanding and predicting plant C turnover response to climate change. A field experiment with ectomycorrhizal fungi (EMF) and nitrogen (N) was established to investigate the effects on allocation of photosynthetic products in Pinus massoniana (Lamb.) seedlings given increased N deposition. Seedlings were subjected to N addition and symbiosis with EMF, and the short-term allocation of a 13C photosynthetic pulse into leaves, branches, stems, roots, and soil was traced. Photosynthetic rate and root respiration were measured. It was found that N addition changed the allocation pattern of photosynthetic products in various organs of P. massoniana. Furthermore, N addition, mycorrhizal symbiosis, and interaction of N and EMF, all increased the amount of C produced by photosynthesis. N application less than 60 kg N hm–1 a–1 could promote the transfer and allocation of photosynthetic products in P. massoniana organs, which peaks at 60 kg N hm–1 a–1, and the highest N treatment began to decrease at 90 kg N hm–1 a–1. EMF inoculation could expand the absorption area of plant roots to obtain more nutrients and synthesize more C and N compounds for promoting the growth of itself and the host plant, improving the net photosynthetic rate and the distribution of C produced by photosynthesis in various organs. This forms a benign C and N cycle, thereby reducing the effect of high N addition on plants. The optimal N addition concentration was 60 kg N hm–1 a–1, and the optimal EMF was Pt, which provides a theoretical basis for inoculating EMF during increasing N deposition in the future climate change scenario. This enables plants to distribute more photosynthetic products to their roots, thus affecting their own C distribution for promoting growth.

Ectomycorrhizal fungi enhance the tolerance of phytotoxicity and cadmium accumulation in oak (Quercus acutissima Carruth.) seedlings: modulation of growth properties and the antioxidant defense responses.

Ectomycorrhizal fungi (EMF), which form symbiotic ectomycorrhiza with tree roots, mediate heavy metal tolerance of host plants. To investigate the roles of EMF in the growth, modulation of oxidative stress, and cadmium (Cd) accumulation and translocation in Quercus acutissima seedlings, ectomycorrhizal seedlings inoculated with Suillus luteus were treated with different Cd concentrations (0.1, and 5 mg kg-1) for 14 days. EMF accelerated seedling growth and Cd accumulation in roots under the highest Cd concentration of 5 mg kg-1. Catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities increased in the leaves of ectomycorrhizal seedlings under the highest Cd concentration. Superoxide dismutase (SOD) trended to increase under both Cd concentrations. Although reduced glutathione (GSH) increased after inoculation of EMF under both Cd concentrations, the release of malondialdehyde increased in the leaves and roots under the highest Cd concentration, indicating that the defense role of EMF in Q. acutissima depends on the Cd concentration. These results indicate that EMF mitigate Cd stress by promoting plant growth and nutrient uptake while modulating the antioxidant system to reduce oxidative stress.

Invasive trees rely more on mycorrhizas, countering the ideal-weed hypothesis.

The ideal-weed hypothesis predicts that invasive plants should be less dependent on mutualisms. However, evidence in favor of or against this hypothesis comes mainly from observational studies. Here, we experimentally tested this hypothesis using a two-factor greenhouse experiment, comparing the seedling growth response of different Pinus species (varying in invasiveness) to ectomycorrhizal fungal inoculation. Most species showed no response until they were 6 mo old, at which point inoculation increased growth between 10 and 260% among the different species. This growth response was higher for species with lower seed mass, higher dispersal ability, higher Z score (a proxy for invasiveness) and higher number of naturalized regions, all of which correspond to higher invasiveness. Our results show that timing is a crucial factor when comparing mycorrhizal dependency of different species. Dependence on mutualistic microorganisms could be part of a strategy in which invasive species produce smaller seeds, in greater number, which can disperse further, but where seedlings are more reliant on mycorrhizas to improve access to water, nutrients, and protection from pathogens. Our results suggest that reliance on mutualisms may enhance, rather than limit, nonnative species in their ability to spread, establish, and colonize.

Screening of ectomycorrhizal and other associated fungi in South African forest nurseries

The South African forestry industry covers approximately 1.3 million hectares and is dependent on exotic pine and eucalypt species. Nursery seedlings are not inoculated with ectomycorrhizal (ECM) fungi. This investigation assessed levels of naturally occurring ECM colonisation of Pinus patula seedlings from 10 different South African forestry nurseries using a grid line intersect method. Fungi from colonised roots were identified using morphological characteristics and Illumina sequencing. Colonisation of seedlings in production nurseries was low (2–21%). Morphologically, the ECM fungi Thelophora terrestris, Suillus sibiricus, and the genera Russula and Pseudotomentella were identified. Molecularly, the ECM fungi T. terrestris, Inocybe jacobi and the genus Sphaerosporella, as well as several other ECM-containing families were identified, along with many saprotrophic/ endophytic fungi belonging to genera such as Penicillium, Ramasonia and Talaromyces. As can be seen, a combination of both molecular and morphological identification techniques are needed as neither is able to give a full picture of the species present in isolation. This study reveals an initial insight into the root microbiome community associated with Pinus patula seedlings, which should be taken into account when inoculation with beneficial microbes is considered. It determined that natural ECM fungal root colonisation levels are very low throughout the South African nurseries investigated, indicating the need for ECM fungal inoculation, which can increase seedling growth, viability and resistance to pathogens.

Identification of candidate genes conferring tolerance to aluminum stress in Pinus massoniana inoculated with ectomycorrhizal fungus

BackgroundPinus massoniana Lamb. is an important afforestation tree species with high economic, ecological and medicinal values. Aluminum (Al) toxicity driven by soil acidification causes dieback of P. massoniana plantations. Previous studies showed that ectomycorrhizal fungi alleviate Al stress damages in Pinus, but the underlying molecular mechanisms and key genes induced by ectomycorrhizal fungi inoculation under Al stress in Pinus have not been explored. Herein, we applied Al stress for 60 days to P. massoniana seedlings inoculated with Suillus luteus (SL) and those non-inoculated. Then, we compared their growth parameters and transcriptome in order to detect candidate genes induced by SL conferring Al tolerance in P. massoniana.ResultOur results showed that SL inoculation confers Al stress tolerance in P. massoniana through improved growth performance, strong antioxidant enzyme activities and reduced malondialdehyde accumulation as compared to non-inoculated seedlings. Transcriptome sequencing further supported these findings as very few genes (51 genes) were transcriptionally altered by Al in SL inoculated plants as compared to non-inoculated plants (2140 genes). We identified three core genes (cox1, cox3 and Nd1) that were strongly up-regulated by Al in the SL inoculated plants but were down-regulated in the non-inoculated plants. We also identified 42 genes specifically regulated by SL inoculated plants under Al stress, which are involved in a wide range of biological processes such as antioxidative response, transporters, hormone signaling and plant pathogen infection responses.ConclusionsAltogether, our data suggest that SL inoculation induces priming of key stress response pathways and triggers specific genes that efficiently alleviate Al stress effects in P. massoniana. The candidate genes resources generated in this study are of utmost importance for functional characterization and molecular studies aiming at improving Al tolerance in plants.

Effects of ectomycorrhizal fungal inoculation on growth and rooting of carob tree (Ceratonia siliqua L.)

The present work investigated the ability of the ectomycorrhizal (ECM) fungi, Pisolithus tinctorius (Pers.) Coker and Couch to improve the growth and root formation in carob seedlings (Ceratonia siliqua L.). Root initiation of hypocotyl cuttings in vitro was enhanced by treatments with different concentrations of either fungal culture filtrate or indole-3-butyric acid (IBA) or in combination. The ectomycorrhization protocol for carob seedlings comprised of three different treatments: in vivo or in vitro inoculation and non-inoculated seedlings. Results obtained after six months of acclimatization showed that inoculation had a positive effect on the growth and rooting of carob plants. Moreover, plants mycorrhized in vitro or in vivo outperformed non-mycorrhized ones in plant height, number of internodes, number of leaves, leaf length, number of roots, maximal root length, and fresh and dry weights of both stems and roots. Indeed, the optimum response was encountered in the seedlings inoculated in vitro. The application of culture filtrate on hypocotyl cuttings revealed significant differences between experimental treatments for the number and length of roots. The best response was recorded in medium containing fungal culture filtrate with 40%. Root formation of hypocotyl cuttings of carob plants was also enhanced by the use of IBA, where the best rooting was manifested in treatment with IBA at a concentration of 2 mg/l. The application of P. tinctorius culture filtrate to the culture medium of hypocotyl cuttings after pretreatment by immersing in IBA was even more effective in improving root formation than the treatment of hypocotyls base with IBA alone. The present study demonstrated the possibility of developing procedures to improve the rooting growth of carob plants by establishing a successful artificial symbiosis with P. tinctorius. Highlights from this work could play a crucial role in arid ecosystem rehabilitation and controlled mycorrhization programs.

Ectomycorrhizal Fungal Inoculation of Sphaerosporella brunnea Significantly Increased Stem Biomass of Salix miyabeana and Decreased Lead, Tin, and Zinc, Soil Concentrations during the Phytoremediation of an Industrial Landfill.

Fast growing, high biomass willows (Salix sp.) have been extensively used for the phytoremediation of trace element-contaminated environments, as they have an extensive root system and they tolerate abiotic stressors such as drought and metal toxicity. Being dual mycorrhizal plants, they can engage single or simultaneous symbiotic associations with both arbuscular mycorrhizal (AM) fungi and ectomycorrhizal (EM) fungi, which can improve overall plant health and growth. The aim of this study was to test the effect of these mycorrhizal fungi on the growth and trace element (TE) extraction potential of willows. A field experiment was carried out where we grew Salix miyabeana clone SX67 on the site of a decommissioned industrial landfill, and inoculated the shrubs with an AM fungus Rhizophagus irregularis, an EM fungus Sphaerosporella brunnea, or a mixture of both. After two growing seasons, the willows inoculated with the EM fungus S. brunnea produced significantly higher biomass. Ba, Cd and Zn were found to be phytoextracted to the aerial plant biomass, where Cd presented the highest bioconcentration factor values in all treatments. Additionally, the plots where the willows received the S. brunnea inoculation showed a significant decrease of Cu, Pb, and Sn soil concentrations. AM fungi inoculation and dual inoculation did not significantly influence biomass production and soil TE levels.

Biological control potential of ectomycorrhizal fungi against Fusarium circinatum on Pinus patula seedlings

ABSTRACT The South Africa forestry industry, covering ∼1.3 million hectares, is dependent on exotic pine and Eucalyptus species. Nursery seedlings are not inoculated with ectomycorrhizal (ECM) fungi or other beneficial microbes. Fusarium circinatum is an economically important pathogen affecting seedling survival. The purpose of this investigation was to assess the effects of ectomycorrhizal fungal inoculation on Pinus patula seedling growth and resistance to the fungal pathogen F. circinatum. Explants from ECM basidiocarps, collected from Pinus stands, were plated onto MMN medium to obtain isolates that were then verified via DNA extraction and PCR amplification and sequencing of the ITS rDNA region. These isolates were identified as Boletus edulis f. reticulatus, Lactarius quieticolor, Suillus granulatus and an unknown Suillus species. P. patula growth in the presence of the pathogen F. circinatum was significantly increased and promoted by the L. quieticolor and Suillus isolates. Preventative inoculation of seedlings in the nursery using these isolates would ensure the production of stronger, healthier plants that would be more resistant to F. circinatum infection, increasing survival in the plantation.

Inoculant production of Pisolithus sp. in submerged culture under agitation

Ectomycorrhizal fungi inoculation can increase the sustainability of planted forests hence the inoculant production should be optimized. The objective of this study was to determine the agitation speed and time of growth for maximum production of mycelium of D216 isolate of Pisolithus sp. The treatments were established by factorial 6x3, being six agitation conditions and three different times (days) of growth. The kinds of agitation used were: Without agitation, gentle manual agitation every two days and orbital agitation speed of 50, 100, 200 and 250 rpm. The times of growth were 14, 21 and 28 days. The mycelium mass was generally single and remained on the surface of the culture medium when grown without agitation or with manual agitation. Spherical mass units formed under agitation and grew submerged. The dry mass production of mycelium of D216 isolate was higher when grown under agitation of 200 rpm, followed by 250 and 100 rpm. For all agitation system, the production of mycelium did not increase after 14 days. The maximum production of mycelium dry mass of D216 isolate of Pisolithus sp. was obtained in the agitation speed of 200 rpm for 14 days. The elevation of the agitation to 250 rpm decreased the mycelium dry mass production of Pisolithus sp. In the absence or in lower agitation speeds, the mycelium dry mass was minimal. Using the ideal speed for maximum mycelium production and adjusting other growing conditions can decrease the risk of contamination, the production time and cost of the inoculant and encourage the use of this biotechnology. Key words: Ectomycorrhizal fungi, inoculation, mycorrhiza, planted forests.

The effects of ectomycorrhizal fungi on heavy metals’ transport in Pinus massoniana and bacteria community in rhizosphere soil in mine tailing area

Pinus massoniana is one of the potential tree species of afforestation in barren mine tailing area, and ectomycorrhizal fungi contributes remarkably to its survival. However, how ectomycorrhizal fungi interacts with Pinus massoniana under heavy metals’ stress is unknown. Two systems (Pinus massoniana inoculated with and without ectomycorrhizal fungi) were designed, and each system contained rhizosphere and non-rhizosphere, while bulk soil was sampled as control. The results showed that treatment of ectomycorrhizal fungi inoculation could obviously improved the absorption of soil moisture, total carbon/total nitrogen and nutrients, while reduced the bulk density and heavy metals of soil when compared with control (p＜0.05). The heavy metals accumulating in plants’ roots with ectomycorrhizal fungi were greater than that without ectomycorrhizal fungi. Conversely, they were lower in shoots with ectomycorrhizal fungi. The bacterial community were affiliated with 23 bacterial phyla, 70 classes, 115 orders, 201 families, and 363 genera. Constrained Principal Coordinate Analysis and redundancy analysis demonstrated that bacterial communities structure in the soil inoculated with or without ectomycorrhizal fungi and bulk soil were distinguishable, but no difference between rhizosphere and non-rhizosphere. The LEfSe analysis showed Acidobacteria, Actinobacteria, and Proteobacteria were the dominant phyla that contributed to the difference among treatments.

Ectomycorrhizal fungi inoculation alleviates simulated acid rain effects on soil ammonia oxidizers and denitrifiers in Masson pine forest.

Acid rain can cause severe effects on soil biota and nutrient biogeochemical cycles in the forest ecosystem, but how plant-symbiotic ectomycorrhizal fungi will modulate the effects remains unknown. Here, we conducted a full factorial field experiment in a Masson pine forest by simultaneously controlling the acidity of the simulated rain (pH 5.6 vs. pH 3.5) and the ectomycorrhizal fungi Pisolithus tinctorius inoculation (non-inoculation vs. inoculation), to investigate the effects on ammonia oxidizers and denitrifiers. After 10 months, compared with the control (rain pH 5.6, and non-inoculation), simulated acid rain (pH 3.5) reduced soil nutrient content, decreased archaeal amoA gene abundance and inhibited denitrification enzyme activity. Also, simulated acid rain altered the community compositions of all the examined functional genes (archaeal amoA, bacterial amoA, nirK, nirS and nosZ). However, inoculation with ectomycorrhizal fungi under acid rain stress recovered soil nutrient content, archaeal amoA gene abundance and denitrification enzyme activity to levels comparable to the control, suggesting that ectomycorrhizal fungi inoculation ameliorates simulated acid rain effects. Taken together, ectomycorrhizal fungi inoculation - potentially through improving soil substrate availability - could alleviate the deleterious effects of acid rain on nitrogen cycling microbes in forest soils.

Does arbuscular and ectomycorrhizal fungal inoculation improve soil aggregate stability? A case study on three tropical species growing in ultramafic Ferralsols

In many tropical regions, soil erosion from anthropogenic activities and subsequent ecological restoration are major issues. Revegetation would be an efficient way to mitigate soil degradation. Some plant and mycorrhizal species are more useful than others for stabilizing soil aggregates and thus reducing erodibility. However, the utility of inoculating plants with mycorrhizas in tropical regions is not clear with regard to the effects on soil aggregate stability. We investigated how plant inoculation with arbuscular (AMF) and ectomycorrhizal (ECM) fungi could improve aggregate stability of ultramafic Ferralsols in New Caledonia. We performed inoculations with local AMF and ECM on three endemic plant species grown in a glasshouse: the sedge Costularia arundinacea (Sol. Ex Vahl) Kük., an AMF host, the shrub Tristaniopsis glauca Brongn. & Gris and the tree Arillastrum gummiferum (Pancher ex Brongn. & Gris) Baill., the latter species are both ECM hosts. We carried out three treatments for each plant species growing in an ultramafic topsoil, (i) control soil with no plants, (ii) non-inoculated plants and (iii) inoculated plants. Inoculation of C. arundinacea was carried out by adding to the topsoil a suspension of mixed AMF spores. T. glauca and A. gummiferum were inoculated with the ECM fungus Pisolithus microcarpus. After 9–10 months, aggregate stability, soil organic carbon, above- and below-ground dry biomass and root traits were measured. Results showed that on ultramafic topsoil, AMF and ECM inoculations significantly increased plant growth, modified roots (especially root length density) and consequently enhanced aggregate stability. Out of the non-inoculated plant species, C. arundinacea had the greatest positive effect on aggregate stability. The association of T. glauca and P. microcarpus was also found to significantly increase aggregate stability. Thus, the inoculation of native plant species with carefully selected mycorrhizal fungi could be an efficient technique to aid in the revegetation of bare slopes.

Parapiptadenia rigida MYCORRHIZATION WITH SPORES OF Scleroderma citrinum

ABSTRACT Ectomycorrhizal fungal inoculation in forestry seedlings aids plant establishment and growth in the field. The objectives of this study were: to determine the mycorrhizal capacity of the ectomycorrhizal fungus Scleroderma citrinum in Parapiptadenia rigida (red angico) seedlings and to evaluate the viability of a mycorrhizal inoculation technique for forest seedlings involving the use of spores. Mature spores were inoculated in the substrate (75% soil and 25% carbonized rice husk), totaling 1.5 grams of fungal spores per liter of substrate. P. rigida seeds were sown in substrates inoculated or not inoculated with fungal spores in presence or absence of Pinus echinata and Eucalyptus citriodora essential oil: not inoculated (T1), inoculated (T2), inoculated more pine essential oil (T3), inoculated more eucalyptus essential oil (T4). Seedlings of Pinus elliottii were used for a positive control of mycorrhizal inoculation (T5) and not inoculated (T6) with fungal spores. At 90 days after sowing, the base stem diameter, height, fresh and dry weight of shoots and roots, percentage of root colonization and Dickson Index were determined. The presence of fungal structures in P. rigida and P. elliottii roots inoculated with S. citrinum spores was observed, demonstrating the occurrence of an ectomycorrhizal association. The application of pine and eucalyptus essential oils in the substrate increased the percentage of ectomycorrhizal colonization in P. rigida seedlings. The addition of S. citrinum mature spores in the substrate used for seedling production is a viable practice for ectomycorrhizal inoculation and it can be used in forest nurseries in controlled mycorrhization programs.

Combination Between Root Pruning and Inoculation Time of Ectomycorrhizal Fungi on Improving Growth of Melinjo (Gnetum gnemon L) Seedling PENGARUH KOMBINASI PEMANGKASAN AKAR DAN WAKTU INOKULASI FUNGI EKTOMIKORIZA TERHADAP PERTUMBUHAN BIBIT MELINJO

The inoculation of ectomycorrhizal fungi that conducted after root pruning could increase the colonization of ectomycorrhizal fungi and growth of melinjo seedling. This research aimed to study the effect of root pruning and inoculation time of ectomycorrhizal fungi on the growth of melinjo seedlings (Gnetum gnemon L). The research were tested in a greenhouse in a completely randomized design with 2 factors for 33 weeks. The first factor is the root pruning (no root pruning as a control, and root pruning 30%). The second factor is the time fungi inoculation (inoculation in the 1st, 2nd, 3rd, 4th and 5th week after root pruning). The combination of root pruning and inoculation time of ectomycorrhizal fungi effected to the growth of melinjo seedlings. The best growth obtained from combination (1) no root pruning and inoculated by ectomycorrhizal fungi in the 1st week, and (2) root pruning 30% and inoculated by ectomycorrhizal fungi in the 3rd week.Key words: ectomycorrhiza, Gnetum gnemon, inoculation time, root pruning, Scleroderma

Inoculation of ectomycorrhizal fungi contributes to the survival of tree seedlings in a copper mine tailing

To advance our understanding of the effects of inoculation with ectomycorrhizal fungi (EMF) on seedling colonization in mine wastelands, we conducted a field experiment in a copper tailing. Six-month-old seedlings of Japanese red pine (Pinus densiflora) and oak (Quercus variabilis) separately inoculated with three EMF species (Pisolithus sp., Cenococcum geophilum, Laccaria laccata) were transplanted to the copper tailing. The survival rates of tree seedlings were monitored monthly, and growth (biomass and height), contents of nutrients and heavy metals (K, P, Ca, Mg, Cu, Zn), and mycorrhizal infection rates of seedlings were determined 6 months after planting. Oak seedlings exhibited higher survival rates than pine seedlings after 6 months of growth on the tailing. EMF inoculations of pine seedlings significantly enhanced their survival, growth, and nutrient uptake. In contrast, EMF inoculations of oak seedlings improved growth only in terms of biomass. Additionally, EMF inoculation caused pine seedlings to accumulate more Cu and Zn in roots compared to non-inoculated seedlings, whereas inoculation inhibited the accumulation of heavy metals in shoots. However, similar results were not observed in oak seedlings. Observations of roots indicated that the rates of mycorrhizal infection of both tree species had dramatically declined at harvest time. In conclusion, ectomycorrhizal symbioses can improve the survival and performance of pine seedlings in mine tailings. The present study provided direct evidence of the importance of EMF inoculation of seedlings to the reforestation of mine wastelands.

Diversity and persistence of ectomycorrhizal fungi and their effect on nursery-inoculated Pinus pinaster in a post-fire plantation in Northern Portugal.

Ectomycorrhizal fungi (ECMF) play an important role in forest ecosystems, often mitigating stress factors and increasing seedling performance. The aim of this study was to investigate the effects of a nursery inoculation on Pinus pinaster growth and on the fungal communities established when reforesting burned areas. Inoculated P. pinaster saplings showed 1.5-fold higher stem height than the non-inoculated controls after a 5 year growth period, suggesting that fungal inoculation could potentiate tree growth in the field. Ordination analysis revealed the presence of different ECMF communities on both plots. Among the nursery-inoculated fungi, Laccaria sp., Rhizopogon sp., Suillus bovinus and Pisolithus sp. were detected on inoculated Pinus saplings on both sampling periods, indicating that they persisted after field establishment. Other fungi were also detected in the inoculated plants. Phialocephala sp. was found on the first assessment, while Terfezia sp. was detected on both sampling periods. Laccaria sp. and Rhizopogon sp. were identified in the control saplings, belonging however to different species than those found in the inoculated plot. Inocybe sp., Thelephora sp. and Paxillus involutus were present on both sampling periods in the non-inoculated plots. The results suggest that ECMF inoculation at nursery stage can benefit plant growth after transplantation to a post-fire site and that the inoculated fungi can persist in the field. This approach has great potential as a biotechnological tool to aid in the reforestation of burned areas.