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In soil ecosystems, rhizobia occupy the rhizosphere of legume roots to form nodules, a process triggered by microbial recognition of specific root-derived signals (i.e. flavonoids). However, soil conditions can limit bacterial motility, restricting signal perception to the area directly influenced by roots. Legumes, like most plants of agricultural interest, associate with arbuscular mycorrhizal fungi, whose hyphae develop extensively in the soil, potentially providing an effective dispersal network for rhizobia. We hypothesized that mycelial networks of arbuscular mycorrhizal fungi play a role in signal transmission and act as a highway, enabling rhizobia to migrate from distant soil to the roots of leguminous plants. Using in vitro and greenhouse microcosm systems, we demonstrated that Rhizophagus irregularis helps Shinorhizobium meliloti to migrate towards the legume Medicago truncatula, triggering nodulation, a mechanism absent without the arbuscular mycorrhizal fungus. Metabolomics analysis revealed eight flavonoids unique to the compartment containing extraradical hyphae of the arbuscular mycorrhizal fungus linked to M. truncatula roots, associated with Sinorhizobium meliloti growth and nod gene expression. Rhizobia plated on the extraradical hyphae connecting two plants (the legume M. truncatula and non-legume Solanum tuberosum) by a common mycelium network, showed preference for the legume, suggesting the chemoattraction by specific signals transported by the fungus connected to the legume. Simultaneously, S. meliloti stimulated the cytoplasmic/protoplasmic flow in the hyphae, likely increasing the release of nutrients and signals. Our results highlight the importance of extraradical hyphae (i.e. the mycorrhizal pathway) of arbuscular mycorrhizal fungi for the migration of rhizobia over long distances to the roots, leading to nodulation., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Background: Microbial culture collections play a key role in taxonomy by studying the diversity of their strains and providing well-characterized biological material to the scientific community for fundamental and applied research. These microbial resource centers thus need to implement new standards in species delineation, including whole-genome sequencing and phylogenomics. In this context, the genomic needs of the Belgian Coordinated Collections of Microorganisms were studied, resulting in the GEN-ERA toolbox. The latter is a unified cluster of bioinformatic workflows dedicated to both bacteria and small eukaryotes (e.g., yeasts)., Findings: This public toolbox allows researchers without a specific training in bioinformatics to perform robust phylogenomic analyses. Hence, it facilitates all steps from genome downloading and quality assessment, including genomic contamination estimation, to tree reconstruction. It also offers workflows for average nucleotide identity comparisons and metabolic modeling., Technical Details: Nextflow workflows are launched by a single command and are available on the GEN-ERA GitHub repository (https://github.com/Lcornet/GENERA). All the workflows are based on Singularity containers to increase reproducibility., Testing: The toolbox was developed for a diversity of microorganisms, including bacteria and fungi. It was further tested on an empirical dataset of 18 (meta)genomes of early branching Cyanobacteria, providing the most up-to-date phylogenomic analysis of the Gloeobacterales order, the first group to diverge in the evolutionary tree of Cyanobacteria., Conclusion: The GEN-ERA toolbox can be used to infer completely reproducible comparative genomic and metabolic analyses on prokaryotes and small eukaryotes. Although designed for routine bioinformatics of culture collections, it can also be used by all researchers interested in microbial taxonomy, as exemplified by our case study on Gloeobacterales., (© The Author(s) 2023. Published by Oxford University Press GigaScience.)

This review highlights new advances in the emerging field of 'Fungi-on-a-Chip' microfluidics for single-cell studies on fungi and discusses several future frontiers, where we envisage microfluidic technology development to be instrumental in aiding our understanding of fungal biology. Fungi, with their enormous diversity, bear essential roles both in nature and our everyday lives. They inhabit a range of ecosystems, such as soil, where they are involved in organic matter degradation and bioremediation processes. More recently, fungi have been recognized as key components of the microbiome in other eukaryotes, such as humans, where they play a fundamental role not only in human pathogenesis, but also likely as commensals. In the food sector, fungi are used either directly or as fermenting agents and are often key players in the biotechnological industry, where they are responsible for the production of both bulk chemicals and antibiotics. Although the macroscopic fruiting bodies are immediately recognizable by most observers, the structure, function, and interactions of fungi with other microbes at the microscopic scale still remain largely hidden. Herein, we shed light on new advances in the emerging field of Fungi-on-a-Chip microfluidic technologies for single-cell studies on fungi. We discuss the development and application of microfluidic tools in the fields of medicine and biotechnology, as well as in-depth biological studies having significance for ecology and general natural processes. Finally, a future perspective is provided, highlighting new frontiers in which microfluidic technology can benefit this field., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Background and Aims: Arbuscular mycorrhizal fungi (AMF) play an important role in plant nutrition and protection against pests and diseases, as well as in soil structuration, nutrient cycling and, generally speaking, in sustainable agriculture, particularly under drought, salinity and low input or organic agriculture. However, little is known about the genetics of the AMF-plant association in tomato. The aim of this study was the genetic analysis of root AMF colonization in tomato via the detection of the quantitative trait loci (QTLs) involved., Methods: A population of 130 recombinant inbred lines derived from the wild species Solanum pimpinellifolium, genotyped for 1899 segregating, non-redundant single nucleotide polymorphisms (SNPs) from the SolCAP tomato panel, was characterized for intensity, frequency and arbuscular abundance of AMF colonization to detect the QTLs involved and to analyse the genes within their peaks (2-2.6 Mbp)., Key Results: The three AMF colonization parameters were highly correlated (0.78-0.97) and the best one, with the highest heritability (0.23), corresponded to colonization intensity. A total of eight QTLs in chromosomes 1, 3, 4, 5, 6, 8, 9 and 10 were detected. Seven of them simultaneously affected intensity and arbuscule abundance. The allele increasing the expression of the trait usually came from the wild parent in accordance with the parental means, and several epistatic interactions were found relevant for breeding purposes. SlCCaMK and SlLYK13 were found among the candidate genes. Carbohydrate transmembrane transporter activity, lipid metabolism and transport, metabolic processes related to nitrogen and phosphate-containing compounds, regulation of carbohydrates, and other biological processes involved in the plant defence were found to be over-represented within the QTL peaks., Conclusions: Intensity is genetically the best morphological measure of tomato root AMF colonization. Wild alleles can improve AMF colonization, and the gene contents of AMF colonization QTLs might be important for explaining the establishment and functioning of the AMF-plant symbiosis., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cooperation is a prevalent phenomenon in nature and how it originates and maintains is a fundamental question in ecology. Many efforts have been made to understand cooperation between individuals in the same species, while the mechanisms enabling cooperation between different species are less understood. Here, we investigated under strict in vitro culture conditions if the exchange of carbon and phosphorus is pivotal to the cooperation between the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis and the phosphate solubilizing bacterium (PSB) Rahnella aquatilis. We observed that fructose exuded by the AMF stimulated the expression of phosphatase genes in the bacterium as well as the rate of phosphatase release into the growth medium by regulating its protein secretory system. The phosphatase activity was subsequently increased, promoting the mineralization of organic phosphorus (i.e., phytate) into inorganic phosphorus, stimulating simultaneously the processes involved in phosphorus uptake by the AMF. Our results demonstrated for the first time that fructose not only is a carbon source, but also plays a role as a signal molecule triggering bacteria-mediated organic phosphorus mineralization processes. These results highlighted the molecular mechanisms by which the hyphal exudates play a role in maintaining the cooperation between AMF and bacteria.

Aims: This study aimed at evaluating the impact of seven plant growth-promoting rhizobacteria (PGPR) on root colonization and life cycle of Rhizophagus irregularis MUCL 41833 when co-entrapped in alginate beads., Methods and Results: Two in vitro experiments were conducted. The first consisted of the immobilization of R. irregularis and seven PGPR isolates into alginate beads to assess the effect of the bacteria on the pre-symbiotic growth of the fungus. In the second experiment, the best performing PGPR from experiment 1 was tested for its ability to promote the symbiotic development of the AMF in potato plantlets from three cultivars. Results showed that only one isolate identified as Pseudomonas plecoglossicida (R-67094) promoted germ tube elongation and hyphal branching of germinated spores during the pre-symbiotic phase of the fungus. This PGPR further promoted the symbiotic development of the AMF in potato plants., Conclusions: The co-entrapment of Ps. plecoglossicida R-67094 and R. irregularis MUCL 41833 in alginate beads improved root colonization by the AMF and its further life cycle under the experimental conditions., Significance and Impact of the Study: Co-entrapment of suitable AMF-PGPR combinations within alginate beads may represent an innovative technology that can be fine-tuned for the development of efficient consortia-based bioformulations., (© 2016 The Society for Applied Microbiology.)

The rhizosphere hosts a rich microflora supporting plant nutrition and health. We examined bacterial rhizosphere microbiota of Solanum tuberosum grown in its center of origin, the Central Andean Highlands, at different vegetation stages and sites at altitudes ranging from 3245 to 4070 m.a.s.l., differing in soil characteristics, climate and the agricultural practices by 454 sequence analysis of 16S rRNA genes. We observed that the taxonomic composition of bacteria repeatedly occurring at particular stages of plant development was almost unaffected by highly diverse environmental conditions. A detailed statistical analysis on the operational taxonomic unit (OTU) level, representing bacterial species, revealed a complex community structure of the rhizosphere. We identified an opportunistic microbiome which comprises OTUs that occur randomly or under specific environmental conditions. In contrast, core microbiome members were found at all sites. The 'stable' component of the core microbiome consisted of few ubiquitous OTUs that were continuously abundant in all samples and vegetation stages, whereas the 'dynamic' component comprised OTUs that were enriched at specific vegetation stages., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Arbuscular mycorrhizal fungi (AMF) are key determinants of plant interactions in ecosystems. Through their effects on competition, they are regulators of the structure of communities. Conversely, the composition of plant assemblages may also influence the AMF colonization dynamics of plant species. Here, we tested under in vitro culture conditions the effects of Medicago truncatula, a highly mycorrhizal plant species, and Silene vulgaris, a weakly mycorrhizal plant species, grown single (monospecies treatments) or in combination (bispecies treatment) on the colonization dynamics of the AMF Rhizophagus irregularis MUCL 43194. The seedlings were placed in a pre-established hyphal network developing from a mature M. truncatula mycorrhizal donor plant. Extraradical mycelium (ERM) and root colonization parameters as well as root morphology were measured over a period of 12 days. An increased ERM length, total root colonization and proportion of arbuscules were noted in the bispecies treatment. Conversely, the bispecies treatment seemed to have no effect on root growth. This study also demonstrated the suitability of the in vitro culture system for studying the interactions between AMF and host plants grown as mono- and bispecies combinations.

Trichoderma sp. is a biocontrol agent active against plant pathogens via mechanisms such as mycoparasitism. Recently, it was demonstrated that Trichoderma harzianum was able to parasitize the mycelium of an arbuscular mycorrhizal (AM) fungus, thus affecting its viability. Here, we question whether this mycoparasitism may reduce the capacity of Glomus sp. to transport phosphorus ((33)P) to its host plant in an in vitro culture system. (33)P was measured in the plant and in the fungal mycelium in the presence/absence of T. harzianum. The viability and metabolic activity of the extraradical mycelium was measured via succinate dehydrogenase and alkaline phosphatase staining. Our study demonstrated an increased uptake of (33)P by the AM fungus in the presence of T. harzianum, possibly related to a stress reaction caused by mycoparasitism. In addition, the disruption of AM extraradical hyphae in the presence of T. harzianum affected the (33)P translocation within the AM fungal mycelium and consequently the transfer of (33)P to the host plant. The effects of T. harzianum on Glomus sp. may thus impact the growth and function of AM fungi and also indirectly plant performance by influencing the source-sink relationship between the two partners of the symbiosis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Aims: This study was performed to explore the compatibility and applicability of plant beneficial micro-organisms (i.e. Trichoderma harzianum MUCL 29707 and Glomus sp. MUCL 41833) co-entrapped in alginate beads., Methods and Results: Spores of Glomus sp. and conidia of T. harzianum were immobilized in alginate beads and the impacts of the saprotrophic fungi on the presymbiotic and symbiotic phase of the arbuscular mycorrhizal (AM) fungi evaluated under strict in vitro culture conditions. Our results demonstrated the capacity of both micro-organisms in combination to regrowth outside the calcium alginate coating. The presence of T. harzianum did not hinder the AM fungal development but rather stimulated its spore production and fitness., Conclusions: The combination of T. harzianum MUCL 29707 with Glomus sp. MUCL 41833 in alginate beads may represent a reliable alternative inoculum formulation for application in sustainable agriculture., Significance and Impact of the Study: The entrapment in the alginate beads of two fungi (i.e. a saprotroph and a symbiont) having beneficial effects on plants represents a promising formulation for the development of inoculants adapted to field application., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Within the rhizosphere, arbuscular mycorrhizal (AM) fungi interact with a cohort of microorganisms, among which is the biological control agent, Trichoderma spp. This fungus parasitizes a wide range of phytopathogenic fungi, a phenomenon also reported in the extraradical mycelium (ERM) of AM fungi. Here, we question whether the mycoparasitism of the ERM could be extended to the intraradical mycelium (IRM), thus representing a pathway for the entry of Trichoderma harzianum within the root. Microcosm experiments allowing interactions between Glomus sp. MUCL 41833 placed in a clade that contains the recently described species Glomus irregulare and T. harzianum were set up under in vitro autotrophic culture conditions using potato as a host. A microscope camera-imaging system, coupled with succinate dehydrogenase staining, was used to assess the mycoparasitism in the ERM and IRM. Trichoderma harzianum colonized the ERM of the AM fungus and spread into the IRM, before exiting into the root cells. Intrahyphal growth of T. harzianum caused protoplasm degradation, decreasing the ERM and IRM viability. ERM of the AM fungus represented a pathway for the entry of T. harzianum into the roots of potato. It further sets off the debate on the susceptibility of the AM fungi of being infected by microorganisms from the rhizosphere.

Arbuscular mycorrhizal (AM) fungi obligatorily depend on carbon (C) resources provided via the plant and therefore fluctuations in C availability may strongly and differently affect AM fungi with different life-history strategies (LHS). In the present study, we examined the effect of repeated defoliation of in vitro grown barrel medic (Medicago truncatula) on the spore and auxiliary cell (AC) production dynamics of a presumed r-strategist (Glomus intraradices) and a presumed K-strategist (Dentiscutata reticulata). Glomus intraradices modulated the production of spores directly to C availability, showing direct investment in reproduction as expected for r-strategists. In contrast, AC production of D. reticulata was not affected after a single defoliation and thus showed higher resistance to fluctuating C levels, as expected for K-strategists. Our results demonstrate that plant defoliation affects the production of extraradical C storage structures of G. intraradices and D. reticulata differently. Our results contribute towards revealing differences in LHS among AM fungal species, a step further towards understanding their community dynamics in natural ecosystems and agroenvironments.

Background: Recent reports suggest that the preoperative use of infliximab (IFX) increases postoperative infectious complications in patients with ulcerative colitis (UC). Therefore, we determined the impact of IFX on postoperative infectious complications., Methods: A consecutive group of 141 UC patients (41% female, median age 39.8 years) undergoing (procto)colectomy was studied. Postoperative infectious complications were compared between 22 patients who received IFX within 12 weeks prior to (procto)colectomy (IFX group) and 119 patients who did not (control group). Short-term infectious complications, consisting of anastomotic leaks, pelvic abscesses, wound infections, and nonsurgical site infections, were recorded within 30 days after primary surgery., Results: At primary surgery there was no significant difference in gender, disease extent, smoking behavior, body mass index, and concomitant medication (including corticosteroids) between the groups. Patients in the IFX group less often underwent restorative proctocolectomy without defunctioning ileostomy (9% versus 34%, P = 0.022), had a significantly shorter median (interquartile range, IQR) disease duration (2.7 [1.2-8.6] versus 5.9 [2.6-13.0] years, P < 0.036) and a significantly higher C-reactive protein level at primary surgery (51.7 [9.9-103.6] versus 19.1 [7.5-42.6] mg/L, P = 0.023). There was no short-term mortality. A moderate-to-high dose of corticosteroids (>or=20 mg methylprednisolone for >or=2 months, odds ratio 5.19 [95% confidence interval [CI]: 1.72-15.66], P = 0.003) and a restorative proctocolectomy without defunctioning ileostomy (odds ratio 6.45 [95% CI: 2.12-19.64], P = 0.001) were independent predictors of short-term postoperative infectious complications., Conclusion: Corticosteroids and a restorative proctocolectomy without defunctioning ileostomy, but not IFX, are associated with an increased risk of short-term postoperative infectious complications in UC.

Carbon transfer between plants via a common extraradical network of arbuscular mycorrhizal (AM) fungal hyphae has been investigated abundantly, but the results remain equivocal. We studied the transfer of carbon through this fungal network, from a Medicago truncatula donor plant to a receiver (1) M. truncatula plant growing under decreased light conditions and (2) M. truncatula seedling. Autotrophic plants were grown in bicompartmented Petri plates, with their root systems physically separated, but linked by the extraradical network of Glomus intraradices. A control Myc-/Nod- M. truncatula plant was inserted in the same compartment as the receiver plant. Following labeling of the donor plant with 13CO2, 13C was recovered in the donor plant shoots and roots, in the extraradical mycelium and in the receiver plant roots. Fatty acid analysis of the receiver's roots further demonstrated 13C enrichment in the fungal-specific lipids, while almost no label was detected in the plant-specific compounds. We conclude that carbon was transferred from the donor to the receiver plant via the AM fungal network, but that the transferred carbon remained within the intraradical AM fungal structures of the receiver's root and was not transferred to the receiver's plant tissues.

Background and Aims: Pouchitis, the most common complication after proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis, has been attributed to altered composition of faecal flora. We investigated the role of antimicrobial and antiglycan antibodies and polymorphisms in microbial pattern recognition receptor genes., Methods: Clinical charts of all 184 patients with ulcerative colitis who underwent IPAA between 1990-2004 were reviewed for pre- and post-operative disease course., Results: Follow-up data were available in 172 patients [67 female, median age at proctocolectomy 39.1 years]. During a median follow-up of 6.7 (interquartile range 3.7-10.5) years, 80 patients (47%) developed at least one episode of pouchitis. Cox proportional-hazard regression identified extra-intestinal manifestations [HR 1.78 (95%CI 1.10-2.88), p=0.020], a GT/TT genotype at Toll-like-receptor-1 S87I [HR 1.64 (1.01-2.66), p=0.047], anti-chitobioside carbohydrate antibodies [HR 2.03 (1.11-3.70), p=0.021] and young age at diagnosis [p=0.003] to be independently associated with pouchitis. Factors associated with chronic pouchitis, diagnosed in 33 patients (19%), were extra-intestinal manifestations [HR 2.45 (1.07-5.62), p=0.034], backwash ileitis [HR 3.15 (1.10-9.00), p=0.032], outer-membrane porin antibodies [HR 2.67 (1.20-5.94), p=0.016] and young age at proctocolectomy [p=0.008]., Conclusions: : The reported association with antibodies and Toll-like-receptor-1 supports the pathophysiological role of the faecal flora in the development of pouchitis.

Background: During the course of their disease, about 30% of patients with ulcerative colitis (UC) will undergo proctocolectomy with ileal pouch-anal anastomosis (IPAA). We evaluated the outcome of IPAA in a Belgian referral center., Methods: Clinical charts were reviewed for pre- and postoperative disease course, functional outcome, and complications in all patients with UC (n = 182) and indeterminate colitis (n = 2) who underwent IPAA in 1990-2004., Results: Follow-up data were available in 173 out of 184 patients (67 female, median age at proctocolectomy 39.0 years). Median functional Oresland score 1 year after IPAA was 3 (range 0-11). Early postoperative complications were seen in 27% of patients. After a median (interquartile range) follow-up of 6.5 (3.4-9.9) years, 35% of patients developed septic and/or obstructive complications. Forty-six percent of patients developed at least 1 episode of pouchitis. Risk factors for pouchitis were the presence of extraintestinal manifestations (odds ratio [OR] 1.92 (1.23-3.01), P = 0.004) and younger age at proctocolectomy (P = 0.004). Chronic pouchitis was present in 33 patients and associated with extraintestinal manifestations (OR 2.93 (1.13-7.62), P = 0.027), backwash ileitis (OR 9.28 (1.71-50.49), P = 0.010), and length of follow-up (P = 0.004). Pouch failure occurred in 5% of patients., Conclusions: Although proctocolectomy with IPAA surgery has a good functional outcome, postoperative complications, especially pouchitis, remain considerable in patients with UC.

The hyphal healing mechanism (HHM) has been shown to differ between Gigasporaceae and Glomeraceae. However, this process has not been considered under (severe) physical stress conditions. Scutellospora reticulata and Glomus clarum strains were cultured monoxenically. The impact of long distance separating cut extremities of hyphae and of multiple injuries within hyphae on the HHM was monitored. For long distances (>5000 microm) separating cut extremities, hyphae healing was observed in half the cases in S. reticulata and was absent in G. clarum. For multiple-injured hyphae, the HHM was always oriented towards the complete recovery of hyphae integrity in S. reticulata, while in G. clarum, the growing hyphal tips (GHTs) could indifferently reconnect cut sections, by-pass cut sections or develop into the environment. Hyphae behaviour under severe physical stress clearly differentiated S. reticulata from G. clarum, suggesting that both fungi have developed different strategies for colony growth to survive under adverse conditions.

An autotrophic culture system was developed for the in vitro mycorrhization of potato plantlets. Roots of micropropagated plantlets were associated to an arbuscular mycorrhizal fungus under in vitro conditions, while shoots developed under open air conditions. Several thousand spores, an extensive extraradical mycelium and an abundant root colonization were obtained. Spores were able to colonize new plantlets under the same conditions. These results support the capacity of the autotrophic culture system to continuously culture arbuscular mycorrhizal fungi and may serve as a powerful tool to investigate various aspects of the symbiosis for which a source-sink relationship and photosynthetic active tissues are necessary.