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.

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.

How does metal soil pollution change the plant mycobiome?

Microorganisms play a key role in plant adaptation to the environment. The aim of this study was to evaluate the effect of toxic metals present in the soil on the biodiversity of plant-related, endophytic mycobiota. The mycobiome of plants and soil from a Zn-Pb heap and a metal-free ruderal area were compared via Illumina sequencing of the ITS1 rDNA. The biodiversity of plants and fungi inhabiting mine dump substrate was lower than that of the metal free site. In the endosphere of Arabidopsis arenosa from the mine dump the number of endophytic fungal taxa was comparable to that in the reference population, but the community structure significantly differed. Agaricomycetes was the most notably limited class of fungi. The results of plant mycobiota evaluation from the field study were verified in terms of the role of toxic metals in plant endophytic fungi community assembly in a reconstruction experiment. The results presented in this study indicate that metal toxicity affects the structure of the plant mycobiota not by changing the pool of microorganisms available in the soil from which the fungal symbionts are recruited but most likely by altering plant and fungi behaviour and the organisms' preferences towards associating in symbiotic relationships.

Iron inactivation by Sporobolomyces ruberrimus and its potential role in plant metal stress protection. An in vitro study.

The endophytic Basidiomycete Sporobolomyces ruberrimus protects its host Arabidopsis arenosa against metal toxicity. Plants inoculated with the fungus yielded more biomass and exhibited significantly fewer stress symptoms in medium mimicking mine dump conditions (medium supplemented with excess of Fe, Zn and Cd). Aside from fine-tuning plant metal homeostasis, the fungus was capable of precipitating Fe in the medium, most likely limiting host exposure to metal toxicity. The precipitated residue was identified by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), X-Ray Diffraction (XRD) and electron microscopy (SEM/TEM) with energy dispersive X-Ray analysis (EDX/SAED) techniques. The performed analyses revealed that the fungus transforms iron into amorphous (oxy)hydroxides and phosphates and immobilizes them in the form of a precipitate changing Fe behaviour in the MSR medium. Moreover, the complexation of free Fe ions by fungi could be obtained by biomolecules such as lipids, proteins, or biosynthesized redox-active molecules.

Endophytic yeast protect plants against metal toxicity by inhibiting plant metal uptake through an ethylene-dependent mechanism.

Toxic metal pollution requires significant adjustments in plant metabolism. Here, we show that the plant microbiota plays an important role in this process. The endophytic Sporobolomyces ruberrimus isolated from a serpentine population of Arabidopsis arenosa protected plants against excess metals. Coculture with its native host and Arabidopsis thaliana inhibited Fe and Ni uptake. It had no effect on host Zn and Cd uptake. Fe uptake inhibition was confirmed in wheat and rape. Our investigations show that, for the metal inhibitory effect, the interference of microorganisms in plant ethylene homeostasis is necessary. Application of an ethylene synthesis inhibitor, as well as loss-of-function mutations in canonical ethylene signalling genes, prevented metal uptake inhibition by the fungus. Coculture with S. ruberrimus significantly changed the expression of Fe homeostasis genes: IRT1, OPT3, OPT6, bHLH38 and bHLH39 in wild-type (WT) A. thaliana. The expression pattern of these genes in WT plants and in the ethylene signalling defective mutants significantly differed and coincided with the plant accumulation phenotype. Most notably, down-regulation of the expression of IRT1 solely in WT was necessary for the inhibition of metal uptake in plants. This study shows that microorganisms optimize plant Fe and Ni uptake by fine-tuning plant metal homeostasis.

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.

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.

Transcriptome Response of Metallicolous and a Non-Metallicolous Ecotypes of Noccaea goesingensis to Nickel Excess.

Root transcriptomic profile was comparatively studied in a serpentine (TM) and a non-metallicolous (NTM) population of Noccaea goesingensis in order to investigate possible features of Ni hyperaccumulation. Both populations were characterised by contrasting Ni tolerance and accumulation capacity. The growth of the TM population was unaffected by metal excess, while the shoot biomass production in the NTM population was significantly lower in the presence of Ni in the culture medium. Nickel concentration was nearly six- and two-fold higher in the shoots than in the roots of the TM and NTM population, respectively. The comparison of root transcriptomes using the RNA-seq method indicated distinct responses to Ni treatment between tested ecotypes. Among differentially expressed genes, the expression of IRT1 and IRT2, encoding metal transporters, was upregulated in the TM population and downregulated/unchanged in the NTM ecotype. Furthermore, differences were observed among ethylene metabolism and response related genes. In the TM population, the expression of genes including ACS7, ACO5, ERF104 and ERF105 was upregulated, while in the NTM population, expression of these genes remained unchanged, thus suggesting a possible regulatory role of this hormone in Ni hyperaccumulation. The present results could serve as a starting point for further studies concerning the plant mechanisms responsible for Ni tolerance and accumulation.

Are Fungal Endophytes Merely Mycorrhizal Copycats? The Role of Fungal Endophytes in the Adaptation of Plants to Metal Toxicity.

The contamination of soil with toxic metals is a worldwide problem, resulting in the disruption of plant vegetation and subsequent crop production. Thus, remediation techniques for contaminated soil and water remain a constant interest of researchers. Phytoremediation, which utilizes plants to remove or stabilize contaminants, is perceived to be a promising strategy. However, phytoremediation's use to date is limited because of constraints associated with such factors as slow plant growth rates or metal toxicity. Microbial-assisted phytoremediation serves as an alternative solution, since the impact of the microbial symbionts on plant growth and stress tolerance has frequently been described. Endophytic fungi occur in almost every plant in the natural environment and contribute to plant growth and tolerance to environmental stress conditions. Although this group of symbiotic fungi was found to form association with a wide range of hosts, including the non-mycorrhizal Brassicaceae metallophytes, their role in the response of plants to metal toxicity has not been thoroughly elucidated to date. This review summarizes the current knowledge regarding the role of endophytic fungi in the tolerance of plants to toxic metals and highlights the similarities and differences between this group of symbiotic fungi and mycorrhizal associations in terms of the survival of the plant during heavy metal stress.

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.

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.

Expansion of a holoparasitic plant, Orobanche lutea (Orobanchaceae), in post-industrial areas - a possible Zn effect.

Industrial waste sites, although extremely difficult to revegetate, may be suitable for rare plants such as Orobanche lutea that are condemned to extinction due to their low ability to compete in their natural habitats. The presence of potentially toxic metals seems to facilitate the expansion of O. lutea (parasitizing Medicago falcata) and was found in hundreds of exemplars per m2 in south Poland and potentially could spread to other localities, causing yield loss in agricultural plants. The main aim of this research was to characterize the interaction between the host, the parasitic plant and symbiotic microbes under different metal concentration in the substratum. The parasite was more common on more polluted soil and when the parasite was connected to the host, potentially toxic metals (Zn, Cd and Pb) were shared by both plants; thus, the content and concentration of these potentially toxic metals in the host were lower than those in plants without parasites. While the performance index (PIABS) of photosynthesis was lower in parasitized plants on control soil, on metal-rich industrial waste soil, PIABS was higher in the parasitized plants than in cases where M. falcata grew alone. This result suggests a role of this parasite in toxicity attenuation, although the biomass of parasitized plants and those growing on polluted sites was lower than that in control sites. In the described case, mycorrhizal colonization and arbuscular richness in M. falcata were even more highly developed on polluted sites than in control ones. The data presented support the hypothesis that the expansion of O. lutea is most likely supported by the increased concentrations of Zn and Cd in areas connected with industrial waste. Although, on industrial wastes the host yield was decreased in the parasite presence, its photosynthetic capacity was even increased.

The Role of Strigolactone in the Cross-Talk Between Arabidopsis thaliana and the Endophytic Fungus Mucor sp.

Over the last years the role of fungal endophytes in plant biology has been extensively studied. A number of species were shown to positively affect plant growth and fitness, thus attempts have been made to utilize these microorganisms in agriculture and phytoremediation. Plant-fungi symbiosis requires multiple metabolic adjustments of both of the interacting organisms. The mechanisms of these adaptations are mostly unknown, however, plant hormones seem to play a central role in this process. The plant hormone strigolactone (SL) was previously shown to activate hyphae branching of mycorrhizal fungi and to negatively affect pathogenic fungi growth. Its role in the plant-endophytic fungi interaction is unknown. The effect of the synthetic SL analog GR24 on the endophytic fungi Mucor sp. growth, respiration, H2O2 production and the activity of antioxidant enzymes was evaluated. We found fungi colony growth rate was decreased in a GR24 concentration dependent manner. Additionally, the fungi accumulated more H2O2 what was accompanied by an altered activity of antioxidant enzymes. Symbiosis with Mucor sp. positively affected Arabidopsis thaliana growth, but SL was necessary for the establishment of the beneficial interaction. A. thaliana biosynthesis mutants max1 and max4, but not the SL signaling mutant max2 did not develop the beneficial phenotype. The negative growth response was correlated with alterations in SA homeostasis and a significant upregulation of genes encoding selected plant defensins. The fungi were also shown to be able to decompose SL in planta and to downregulate the expression of SL biosynthesis genes. Additionally, we have shown that GR24 treatment with a dose of 1 µM activates the production of SA in A. thaliana. The results presented here provide evidence for a role of SL in the plant-endophyte cross-talk during the mutualistic interaction between Arabidopsis thaliana and Mucor sp.