Bacteria Associated with Spores of Arbuscular Mycorrhizal Fungi Improve the Effectiveness of Fungal Inocula for Red Raspberry Biotization.

Intensive crop production leads to the disruption of the symbiosis between plants and their associated microorganisms, resulting in suboptimal plant productivity and lower yield quality. Therefore, it is necessary to improve existing methods and explore modern, environmentally friendly approaches to crop production. One of these methods is biotization, which involves the inoculation of plants with appropriately selected symbiotic microorganisms which play a beneficial role in plant adaptation to the environment. In this study, we tested the possibility of using a multi-microorganismal inoculum composed of arbuscular mycorrhizal fungi (AMF) and AMF spore-associated bacteria for biotization of the red raspberry. Bacteria were isolated from the spores of AMF, and their plant growth-promoting properties were tested. AMF inocula were supplemented with selected bacterial strains to investigate their effect on the growth and vitality of the raspberry. The investigations were carried out in the laboratory and on a semi-industrial scale in a polytunnel where commercial production of seedlings is carried out. In the semi-industrial experiment, we tested the growth parameters of plants and physiological response of the plant to temporary water shortage. We isolated over fifty strains of bacteria associated with spores of AMF. Only part of them showed plant growth-promoting properties, and six of these (belonging to the Paenibacillus genus) were used for the inoculum. AMF inoculation and co-inoculation of AMF and bacteria isolated from AMF spores improved plant growth and vitality in both experimental setups. Plant dry weight was improved by 70%, and selected chlorophyll fluorescence parameters (the contribution of light to primary photochemistry and fraction of reaction centre chlorophyll per chlorophyll of the antennae) were increased. The inoculum improved carbon assimilation, photosynthetic rate, stomatal conductance and transpiration after temporary water shortage. Raspberry biotization with AMF and bacteria associated with spores has potential applications in horticulture where ecological methods based on plant microorganism interaction are in demand.

Biological control of predatory fungi inhabiting activated sludge in wastewater treatment.

The unfavorable phenomenon of activated sludge bulking that occurs in sewage treatment plants (WWTPs) is caused by the over-proliferation of filamentous bacteria that should be limited by the Lecane rotifers that feed on them; however, predatory, rotiferovorous fungi that often inhabit WWTPs pose a real threat to these organisms. To solve this problem, we investigated the interaction of the fungus Clonostachys rosea, which is a known Biological Control Agent (BCA) and the predacious Zoophagus sp. in simplified laboratory culture conditions. The presence of C. rosea in the cultures reduced the number of active traps, thus translating into a much smaller number of rotifers being caught. The mycelium of C. rosea was labeled with a red fluorescent protein (RFP). The life cycle of C. rosea that were attacking Zoophagus sp. (hunting for rotifers) is described. C. rosea spores germinate into single-celled forms and penetrate the interior of the Zoophagus mycelium where they feed on the cytoplasm. Then is the mycelium produced abundantly and forms conidiophores. This type of life strategy has not been known before. The obtained results demonstrated the potential of C. rosea as a BCA that can be used to protect rotifers in the event of an infection of activated sludge by the predatory fungi that threaten the rotifer population.

Biotization of highbush blueberry with ericoid mycorrhizal and endophytic fungi improves plant growth and vitality.

Ecological methods are becoming increasingly popular. One of these methods is plant biotization. In our paper, we focus on selection of Vaccinium corymbosum hairy root-inhabiting fungi for plant growth promotion in a single microorganism inoculation setup and then composed a multiorganismal inoculum enriched with a representative of another group of fungi, leaf endophytes. The hairy roots of V. corymbosum hosted 13 fungal taxa. In single inoculation of the plant with fungal strains, the most beneficial for plant growth were Oidiodendron maius and Phialocephala fortinii. Additional inoculation of the plants with three root symbiotic fungi (O. maius, Hymenoscyphus sp. and P. fortinii) and with the endophytic fungus Xylaria sp. increased plant height in laboratory experiments. On a semi-industrial scale, inoculation improved plant biomass and vitality. Therefore, the amendment of root-associated fungal communities with a mixture of ericoid mycorrhizal and endophytic fungi may represent an alternative to conventional fertilization and pesticide application in large-scale blueberry production. KEY POINTS: • O. maius and P. fortinii significantly stimulated V. corymbosum growth in a single inoculation. • Multimicroorganismal inoculum increased plant biomass and vitality. • Blueberry biotization with ericoid and endophytic fungi is recommended.

Extraordinary Multi-Organismal Interactions Involving Bacteriophages, Bacteria, Fungi, and Rotifers: Quadruple Microbial Trophic Network in Water Droplets.

Our observations of predatory fungi trapping rotifers in activated sludge and laboratory culture allowed us to discover a complicated trophic network that includes predatory fungi armed with bacteria and bacteriophages and the rotifers they prey on. Such a network seems to be common in various habitats, although it remains mostly unknown due to its microscopic size. In this study, we isolated and identified fungi and bacteria from activated sludge. We also noticed abundant, virus-like particles in the environment. The fungus developed absorptive hyphae within the prey. The bacteria showed the ability to enter and exit from the hyphae (e.g., from the traps into the caught prey). Our observations indicate that the bacteria and the fungus share nutrients obtained from the rotifer. To narrow the range of bacterial strains isolated from the mycelium, the effects of bacteria supernatants and lysed bacteria were studied. Bacteria isolated from the fungus were capable of immobilizing the rotifer. The strongest negative effect on rotifer mobility was shown by a mixture of Bacillus sp. and Stenotrophomonas maltophilia. The involvement of bacteriophages in rotifer hunting was demonstrated based on molecular analyses and was discussed. The described case seems to be an extraordinary quadruple microbiological puzzle that has not been described and is still far from being understood.

Acclimation of the photosynthetic apparatus and alterations in sugar metabolism in response to inoculation with endophytic fungi.

The role of an endophytic Zygomycete Mucor sp. in growth promotion and adaptation of the photosynthetic apparatus to increased energy demands of its hosts Arabidopsis arenosa and Arabidopsis thaliana was evaluated. Inoculation with the fungus improved the water use efficiency of the plants and allowed for them to utilize incident light for photochemistry more effectively by upregulating the expression of several photosystem I- and II-related genes and their respective proteins, proteins involved in light harvesting in PSII and PSI and carbon assimilation. This effect was independent of the ability of the plants to acquire nutrients from the soil. We hypothesize that the accelerated growth of the symbiotic plants resulted from an increase in their demand for carbohydrates and carbohydrate turnover (sink strength) that triggered a simultaneous upregulation of carbon assimilation. Arabidopsis plants inoculated with Mucor sp. exhibited upregulated expression in several genes encoding proteins involved in carbohydrate catabolism, sugar transport, and smaller starch grains that indicate a significant upregulation of carbohydrate metabolism.

Mucor sp.-An endophyte of Brassicaceae capable of surviving in toxic metal-rich sites.

The aim of this study was to assess the biodiversity of endophytic fungi from Arabidopsis arenosa growing on a post mining waste dump and to evaluate their role in plant adaptation to metal toxicity. Severeal of the fungi were beneficial for the plant. Among them, a fungus belonging to the Mucor genus, was found to interact with a broad range of plants, including Brassicaceae metallophytes. Mucor sp. was shown to be highly tolerant to elevated levels of Zn, Cd, and Pb and to accelerate plant-host growth under either toxic-metal stress or control conditions. When inoculated with Mucor sp., A. arenosa under toxic-metal stress acquired more N and showed significantly down-regulated catalase activity, which suggests suppression of toxic-metal-induced oxidative stress. We used the model plant-A. thaliana to evaluate the dynamics of plant-tissue colonization by the fungus as monitored with qPCR and to analyze the host's transcriptome response during early stages of the interaction. The results revealed the induction of a plant-defense and stress-related response on the 5th day of co-culture, which was in accord with the decrease of fungal abundance in shoots on the 6th day of interaction. Presented results demonstrate the importance of endophytic fungi in plant toxic-metal tolerance.

Does co-inoculation of Lactuca serriola with endophytic and arbuscular mycorrhizal fungi improve plant growth in a polluted environment?

Phytoremediation of polluted sites can be improved by co-inoculation with mycorrhizal and endophytic fungi. In this study, the effects of single- and co-inoculation of Lactuca serriola with an arbuscular mycorrhizal (AM) fungus, Rhizoglomus intraradices, and endophytic fungi, Mucor sp. or Trichoderma asperellum, on plant growth, vitality, toxic metal accumulation, sesquiterpene lactone production and flavonoid concentration in the presence of toxic metals were evaluated. Inoculation with the AM fungus increased biomass yield of the plants grown on non-polluted and polluted substrate. Co-inoculation with the AM fungus and Mucor sp. resulted in increased biomass yield of plants cultivated on the polluted substrate, whereas co-inoculation with T. asperellum and the AM fungus increased plant biomass on the non-polluted substrate. In the presence of Mucor sp., mycorrhizal colonization and arbuscule richness were increased in the non-polluted substrate. Co-inoculation with the AM fungus and Mucor sp. increased Zn concentration in leaves and roots. The concentration of sesquiterpene lactones in plant leaves was decreased by AM fungus inoculation in both substrates. Despite enhanced host plant costs caused by maintaining symbiosis with numerous microorganisms, interaction of wild lettuce with both mycorrhizal and endophytic fungi was more beneficial than that with a single fungus. The study shows the potential of double inoculation in unfavourable environments, including agricultural areas and toxic metal-polluted areas.

Interactions of arbuscular mycorrhizal and endophytic fungi improve seedling survival and growth in post-mining waste.

The impact of fungal endophytes and the modulating role of arbuscular mycorrhizal fungi (AMF) on the vitality of Verbascum lychnitis, grown in the laboratory in a substratum from a post-mining waste dump was investigated. We report that inoculation with a single endophyte negatively affected the survival rate and biomass production of most of the plant-endophyte consortia examined. The introduction of arbuscular mycorrhiza fungi into this setup (dual inoculation) had a beneficial effect on both biomass yield and survivability. V. lychnitis co-inoculated with AMF and Cochliobolus sativus, Diaporthe sp., and Phoma exigua var. exigua yielded the highest biomass, exceeding the growth rate of both non-inoculated and AMF plants. AMF significantly improved the photosynthesis rates of the plant-endophyte consortia, which were negatively affected by inoculation with single endophytes. The abundance of PsbC, a photosystem II core protein previously shown to be upregulated in plants colonized by Epichloe typhina, exhibited a significant increase when the negative effect of the fungal endophyte was attenuated by AMF.

Interplay between carotenoids, hemoproteins and the "life band" origin studied in live Rhodotorula mucilaginosa cells by means of Raman microimaging.

Raman microimaging of live Rhodotorula mucilaginosa cells, cultured under aerobic and anaerobic conditions, showed striking differences in the composition and distribution of cell components. The analysis of these differences and recovery of oxidative phosphorylation upon environmental changes enabled the interrelation of carotenoids, hemoproteins and the unknown species considered as the "Raman signature of life".

Microscopic processes ruling the bioavailability of Zn to roots of Euphorbia pithyusa L. pioneer plant.

Euphorbia pithyusa L. was used in a plant growth-promoting assisted field trial experiment. To unravel the microscopic processes at the interface, thin slices of E. pithyusa roots were investigated by micro-X-ray fluorescence mapping. Roots and rhizosphere materials were examined by X-ray absorption spectroscopy at the Zn K-edge, X-ray diffraction, and scanning electron microscopy. Results indicate some features common to all the investigated samples. (i) In the rhizosphere of E. pithyusa, Zn was found to exist in different phases. (ii) Si and Al are mainly concentrated in a rim at the epidermis of the roots. (iii) Zn is mostly stored in root epidermis and does not appear to be coordinated to organic molecules but mainly occurs in mineral phases such as Zn silicates. We interpreted that roots of E. pithyusa significantly promote mineral evolution in the rhizosphere. Concomitantly, the plant uses Si and Al extracted by soil minerals to build a biomineralization rim, which can capture Zn. This Zn silicate biomineralization has relevant implications for phytoremediation techniques and for further biotechnology development, which can be better designed and developed after specific knowledge of molecular processes ruling mineral evolution and biomineralization processes has been gained.

Mycology: The overlooked but obvious process of fungal-induced fossil biomineralization.

The ability of fungi to mineralize substrates is a common but underestimated phenomenon. Now, researchers have demonstrated the usefulness of this process in fossilization by documenting the formation of hydroxyapatite nanofibres. This opens new avenues in palaeomycology and palaeontology.

Fungal symbionts impact cyanobacterial biofilm durability and photosynthetic efficiency.

Cyanobacteria contribute to over 25% of the world's net primary photosynthetic production and are pivotal in mitigating greenhouse gas emissions.1 This study unveils a previously unobserved symbiotic relationship between benthic cyanobacteria and fungi that have also adapted to life as a plant endophyte. The interaction suggests an initial phase of lichenization. We isolated Leptolyngbya frigida from the Naracauli stream, which emanates from abandoned Zn industrial waste in Sardinia. Seasonally, L. frigida participates in a biomineralization processes, mitigating the Zn transfer to rivers and, subsequently, the sea.2,3,4L. frigida is a benthic cyanobacterium that establishes a biofilm on the stream bed. Notably, the area predominantly features Juncus acutus. From these roots, endophytic fungi were predominantly isolated as Clonostachys rosea, a fungus recognized for its biocontrol capabilities against plant pathogens. An intriguing observation was made when L. frigida was cultured with C. rosea on a low-carbohydrate agar medium: the fungal mycelium transformed into wall-less forms, a phenomenon not documented previously. In liquid environments, the resulting biofilm first settled at the container's bottom. Even upon rising to the surface, this biofilm remained pigment rich. Concurrently, a secondary biofilm began its formation at the bottom. These fungal-integrated biofilms displayed enhanced resilience and superior photosynthetic performance compared to those without fungal presence. Moreover, the symbiotic relationship significantly amplified O2 emission and CO2 sequestration by the biofilm.

Hypericin and pseudohypericin concentrations of a valuable medicinal plant Hypericum perforatum L. are enhanced by arbuscular mycorrhizal fungi.

Hypericum perforatum L. (St. John's-wort, Hypericaceae) is a valuable medicinal plant species cultivated for pharmaceutical purposes. Although the chemical composition and pharmacological activities of H. perforatum have been well studied, no data are available concerning the influence of arbuscular mycorrhizal fungi (AMF) on this important herb. A laboratory experiment was therefore conducted in order to test three AMF inocula on H. perforatum with a view to show whether AMF could influence plant vitality (biomass and photosynthetic activity) and the production of the most valuable secondary metabolites, namely anthraquinone derivatives (hypericin and pseudohypericin) as well as the prenylated phloroglucinol-hyperforin. The following treatments were prepared: (1) control-sterile soil without AMF inoculation, (2) Rhizophagus intraradices (syn. Glomus intraradices), (3) Funneliformis mosseae (syn. Glomus mosseae), and (4) an AMF Mix which contained: Funneliformis constrictum (syn. Glomus constrictum), Funneliformis geosporum (syn. Glomus geosporum), F. mosseae, and R. intraradices. The application of R. intraradices inoculum resulted in the highest mycorrhizal colonization, whereas the lowest values of mycorrhizal parameters were detected in the AMF Mix. There were no statistically significant differences in H. perforatum shoot mass in any of the treatments. However, we found AMF species specificity in the stimulation of H. perforatum photosynthetic activity and the production of secondary metabolites. Inoculation with the AMF Mix resulted in higher photosynthetic performance index (PI(total)) values in comparison to all the other treatments. The plants inoculated with R. intraradices and the AMF Mix were characterized by a higher concentration of hypericin and pseudohypericin in the shoots. However, no differences in the content of these metabolites were detected after the application of F. mosseae. In the case of hyperforin, no significant differences were found between the control plants and those inoculated with any of the AMF applied. The enhanced content of anthraquinone derivatives and, at the same time, better plant vitality suggest that the improved production of these metabolites was a result of the positive effect of the applied AMF strains on H. perforatum. This could be due to improved mineral nutrition or to AMF-induced changes in the phytohormonal balance. Our results are promising from the biotechnological point of view, i.e. the future inoculation of H. perforatum with AMF in order to improve the quality of medicinal plant raw material obtained from cultivation.

Effects of genetic modifications to flax (Linum usitatissimum) on arbuscular mycorrhiza and plant performance.

Although arbuscular mycorrhizal fungi (AMF) are known for their positive effect on flax growth, the impact of genetic manipulation in this crop on arbuscular mycorrhiza and plant performance was assessed for the first time. Five types of transgenic flax that were generated to improve fiber quality and resistance to pathogens, through increased levels of either phenylpropanoids (W92.40), glycosyltransferase (GT4, GT5), or PR2 beta-1,3-glucanase (B14) or produce polyhydroxybutyrate (M50), were used. Introduced genetic modifications did not change the degree of mycorrhizal colonization as compared to parent cultivars Linola and Nike. Arbuscules were well developed in each tested transgenic type (except M50). In two lines (W92.40 and B14), a higher abundance of arbuscules was observed when compared to control, untransformed flax plants. However, in some cases (W92.40, GT4, GT5, and B14 Md), the mycorrhizal dependency for biomass production of transgenic plants was slightly lower when compared to the original cultivars. No significant influence of mycorrhiza on the photosynthetic activity of transformed lines was found, but in most cases P concentration in mycorrhizal plants remained higher than in nonmycorrhizal ones. The transformed flax lines meet the demands for better quality of fiber and higher resistance to pathogens, without significantly influencing the interaction with AMF.

In situ Raman imaging of astaxanthin in a single microalgal cell.

Raman imaging is shown to be a highly selective and sensitive method of studying in situ and in vivo astaxanthin distribution, concentration and molecular structure in the cyst form of the unicellular microalgae Haematococcus pluvialis.

Mycology: Protein Control of Fungal Nanoparticle Formation.

The ability of fungi to immobilize metals is an extraordinary phenomenon. Now, a protein involved in the formation of copper carbonate nanoparticles has been identified, and suggests that the process is under the control of the fungus, opening new avenues for the synthesis of nanomaterials.

Cooling effect of fungal stromata in the Dactylis-Epichloë-Botanophila symbiosis.

The stromata of Epichloë fungi are structures covering part of the stem of grasses. Under the fungal layer, still green tissues of the plant survive, although the development of the new leaves is inhibited. Stromata are the places where conidia and ascospores develop. Also, here Botanophila flies dine on mycelium, lay the eggs, defecate, and the larvae develop. The interaction of the three symbionts was analyzed concerning the organisms' adaptation to understand the differences in physiology and ecology of this microenvironment that support stable symbiosis spreading presently in Europe since the beginning of the XXI century. For analysis of the infrared radiation emitted by stromata, a high-resolution infrared camera FLIR E50 was used. The visualization of stromata temperature profiles was shown in the form of pseudo-colored (false) infrared images. The 13C discrimination was used to characterize photosynthesis of the plant tissue enclosed within the stromata. The stromata had a substantially lower temperature than the green plant tissues. The difference reached ~5.6°C during midday hours, whereas it was smaller in the evening, reaching only ~3.6°C. The mycelium of Epichloë cultivated on agar showed about 2°C lower temperature in comparison to the surrounding. The plant tissues enclosed within the stroma were photosynthetically active, although this activity was of phosphoenolpyruvate carboxylase (PEPC) type and less involved in heat dissipation during the day. The stromata, built by fungal hyphae, on which fungal reproductive structures develop, form a cool shelter. This shelter provides a place for the larvae of Botanophila flies.

Phytohormone based biostimulant combined with plant growth promoting endophytic fungus enhances Ni phytoextraction of Noccaea goesingensis.

To improve the efficiency of Ni phytoextraction, the metal hyperaccumulator N. goesingensis was subject to treatment with a combination of a Ni uptake stimulating microorganism and the commercially available, IAA- based biostimulating seaweed extract - Kelpak. Additionally, we compared the plant growth promoting and Ni uptake capabilities of the two biofertilizers. Treatment with the Kelpak alone had no significant effect on plant growth or Ni accumulation. Inoculation of N. goesingensis with Phomopsis columnaris significantly improved the biomass of the hyperaccumulating plant and Ni yield per plant and improved several plant biometric features such as fresh and dry weight and several others related to leaf and root size. However, the combination of the two treatments yielded the best results; plants treated with the two growth promoting agents yielded 85% more biomass compared to not treated plants and accumulated 48% more Ni per plant. To verify plant inoculation with the fungus we generated a GFP expressing strain of P. columnaris and visualized the fungus in both plant leaves and roots. To trace the development of the fungus in planta and to evaluate the effect of biostimulant treatment on mycelium development fungal translational elongation factor 1α (tef1α) DNA was quantified with qPCR. Upon biofertilizer the abundance P. columnaris in plant leaves increased nearly 5-fold. The utilization of plant growth stimulating microorganisms, endophytic fungi in particular, can significantly improve Ni phytoextraction in hyperaccumulator N. goesingensis.

The effect of endophytic fungi on growth and nickel accumulation in Noccaea hyperaccumulators.

The role of endophytic fungi isolated from different populations of European Ni hyperaccumulators was investigated in regard to the microorganisms' ability to enhance the hyperaccumulation of Ni in Noccaea caerulescens. Effects of particular species of endophytic fungi on adaptation of N. caerulescens to excess Ni were tested by co-cultivation with single strains of the fungi. Seven of these had a positive effect on plant biomass production, whereas two of the tested species inhibited plant growth; biomass production of inoculated plants was significantly different compared to non-inoculated control. Inoculation with six fungal strains: Embellisia thlaspis, Pyrenochaeta cava, Phomopsis columnaris, Plectosphaerella cucumerina, Cladosporium cladosporioides and Alternaria sp. stimulated the plant to uptake and accumulate more Ni in both roots and shoots, compared to non-inoculated control. P. columnaris was isolated from all plant species sampled. Strains isolated from Noccaea caerulescens and Noccaea goesingensis increased Ni root and shoot accumulation of their native hosts (compared to non-inoculated control). Inoculation of different populations of Noccaea with P. columnaris of foreign origin did not cause its host to accumulate more Ni, with the exception of the Ni-unadapted ecotype of N. goesingensis. Inoculation with P. columnaris from N. caerulescens significantly improved Ni uptake, but the effect of the fungus was not as prominent as in the case of N. caerulescens. By comparing the transcriptomes of N. caerulescens and N. goesingensis from Flatz inoculated with P. columnaris, we showed that enhanced uptake and accumulation of Ni in the plants is accompanied by an upregulation of several genes mainly involved in plant stress protection and metal uptake and compartmentation.

The potential role of arbuscular mycorrhizal fungi in protecting endangered plants and habitats.

Ecosystems worldwide are threatened with the extinction of plants and, at the same time, invasion by new species. Plant invasiveness and loss of species can be caused by similar but opposing pressures on the community structures. Arbuscular mycorrhizal fungi (AMF) can have multiple positive effects on plant growth, productivity, health, and stress relief. Many endangered species live in symbiosis with AMF. However, the list of the International Union for Conservation of Nature and Natural Resources (IUCN Red List of Threatened Species) indicates that the mycorrhizal status of most of the threatened species has not been assessed. Rare plants often occur in specialized and also endangered habitats and might utilize specialized or unique AMF. The specificity of any endangered plant to its AMF population has not been investigated. Because most of the current AMF isolates that are available colonize a broad range of plant species, selected inocula could be used to promote growth of endangered plants before the proper and more effective indigenous AMF are characterized. Application of AMF in field sites to protect endangered plants is hardly feasible due to the complexity of plant community structures and the large amount of fungal inocula needed. Endangered plants could, however, be grown as greenhouse cultures together with appropriate fungi, and, at the relevant developmental stage, they could be re-planted into native sites to prevent extinction and to preserve plant community ecology.