Fungal holobionts as blueprints for synthetic endosymbiotic systems.

Rhizopus microsporus is an example of a fungal holobiont. Strains of this species can harbor bacterial and viral endosymbionts inherited by the next generation. These microbial allies increase pathogenicity and defense and control asexual and sexual reproduction.

Genome-Scale Model of Rhizopus microsporus: Metabolic integration of a fungal holobiont with its bacterial and viral endosymbionts.

Rhizopus microsporus often lives in association with bacterial and viral symbionts that alter its biology. This fungal model represents an example of the complex interactions established among diverse organisms in functional holobionts. We constructed a Genome-Scale Model (GSM) of the fungal-bacterial-viral holobiont (iHol). We employed a constraint-based method to calculate the metabolic fluxes to decipher the metabolic interactions of the symbionts with their host. Our computational analyses of iHol simulate the holobiont's growth and the production of the toxin rhizoxin. Analyses of the calculated fluxes between R. microsporus in symbiotic (iHol) versus asymbiotic conditions suggest that changes in the lipid and nucleotide metabolism of the host are necessary for the functionality of the holobiont. Glycerol plays a pivotal role in the fungal-bacterial metabolic interaction, as its production does not compromise fungal growth, and Mycetohabitans bacteria can efficiently consume it. Narnavirus RmNV-20S and RmNV-23S affected the nucleotide metabolism without impacting the fungal-bacterial symbiosis. Our analyses highlighted the metabolic stability of Mycetohabitans throughout its co-evolution with the fungal host. We also predicted changes in reactions of the bacterial metabolism required for the active production of rhizoxin. This iHol is the first GSM of a fungal holobiont.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

The Rhizopus Holobiont: A Model to Decipher Fungal-Bacterial-Viral Symbioses.

Rhizopus microsporus is an early-diverging fungal species that inhabits the soil, is used for the fermentation of diverse Asian and African foods, and can be a pathogen of plants, animals, and humans.Toxin-producing strains of R. microsporus live in symbiosis with Gram-negative betaproteobacteria from the genus Mycetohabitans (Burkholderia sensu lato). These bacterial endosymbionts increase the metabolic plasticity of the fungal holobiont by producing the "mycotoxins," control their asexual reproduction, and influence their sexual success. Recently, we identified two viruses of the genus Narnavirus in some R. microsporus strains that harbor Mycetohabitans. By eliminating bacteria and/or viruses from host R. microsporus strains, we have been able to study the role of these symbionts in fungal biology. Remarkably, the absence of these bacterial and viral symbionts decreases sexual reproduction. In this chapter, the method developed to eliminate and genotype the Narnavirus RmNV-20S and RmNV-23S in R. microsporus is described in detail.

Symbiotic and toxinogenic Rhizopus spp. isolated from soils of different papaya producing regions in Mexico.

Mucoralean fungi from the genus Rhizopus are common inhabitants of terrestrial ecosystems, being some pathogens of animals and plants. In this study, we analyzed the symbiotic and toxinogenic potential of Rhizopus species derived from agricultural soils dedicated to the production of papaya (Carica papaya L.) in Mexico. Four representative strains of soil-derived Rhizopus spp. were analyzed employing molecular, microscopic, and metabolic methods. The ITS phylogenies identified the fungi as Rhizopus microsporus HP499, Rhizopus delemar HP475 and HP479, and Rhizopus homothallicus HP487. We discovered that R. microsporus HP499 and R. delemar HP475 harbor similar endofungal bacterial symbionts that belong to the genus Mycetohabitans (Burkholderia sensu lato) and that none of the four fungi were associated with Narnavirus RmNV-20S and RmNV-23S. Intriguingly, the interaction between R. delemar - Mycetohabitans showed different phenotypes from known R. microsporus - Mycetohabitans symbioses. Elimination of bacteria in R. delemar HP475 did not cause a detrimental effect on fungal growth or asexual reproduction. Moreover, metabolic and molecular analyses confirmed that, unlike symbiotic R. microsporus HP499, R. delemar HP475 does not produce rhizoxin, one of the best-characterized toxins produced by Mycetohabitans spp. The rhizoxin (rhi) biosynthetic gene cluster seems absent in this symbiotic bacterium. Our study highlights that the symbioses between Rhizopus and Mycetohabitans are more diverse than anticipated. Our findings contribute to expanding our understanding of the role bacterial symbionts have in the pathogenicity, biology and evolution of Mucorales.

Distinctive prokaryotic microbiomes in sympatric plant roots from a Yucatan cenote.

OBJECTIVE: Cenotes are flooded caves in Mexico's Yucatan peninsula. Many cenotes are interconnected in an underground network of pools and streams forming a vast belowground aquifer across most of the peninsula. Many plants in the peninsula grow roots that reach the cenotes water and live submerged in conditions similar to hydroponics. Our objective was to study the microbial community associated with these submerged roots of the Sac Actun cenote. We accomplished this objective by profiling the root prokaryotic community using 16S rRNA gene amplification and sequencing. RESULTS: We identified plant species by DNA barcoding the total genomic DNA of each root. We found a distinctive composition of the root and water bacterial and archaeal communities. Prokaryotic diversity was higher in all plant roots than in the surrounding freshwater, suggesting that plants in the cenotes may attract and select microorganisms from soil and freshwater, and may also harbor vertically transmitted lineages. The reported data are of interest for studies targeting biodiversity in general and root-microbial ecological interactions specifically.

Narnaviruses: novel players in fungal-bacterial symbioses.

Rhizopus microsporus is an early-diverging fungal species with importance in ecology, agriculture, food production, and public health. Pathogenic strains of R. microsporus harbor an intracellular bacterial symbiont, Mycetohabitans (formerly named Burkholderia). This vertically transmitted bacterial symbiont is responsible for the production of toxins crucial to the pathogenicity of Rhizopus and remarkably also for fungal reproduction. Here we show that R. microsporus can live not only in symbiosis with bacteria but also with two viral members of the genus Narnavirus. Our experiments revealed that both viruses replicated similarly in the growth conditions we tested. Viral copies were affected by the developmental stage of the fungus, the substrate, and the presence or absence of Mycetohabitans. Absolute quantification of narnaviruses in isolated asexual sporangiospores and sexual zygospores indicates their vertical transmission. By curing R. microsporus of its viral and bacterial symbionts and reinfecting bacteria to reestablish symbiosis, we demonstrate that these viruses affect fungal biology. Narnaviruses decrease asexual reproduction, but together with Mycetohabitans, are required for sexual reproductive success. This fungal-bacterial-viral system represents an outstanding model to investigate three-way microbial symbioses and their evolution.

Forest tree associated bacteria for potential biological control of Fusarium solani and of Fusarium kuroshium, causal agent of Fusarium dieback.

Although the use of crop-associated bacteria as biological control agents of fungal diseases has gained increasing interest, the biotechnological potential of forest tree-associated microbes and their natural products has scarcely been investigated. The objective of this study was to identify bacteria or bacterial products with antagonistic activity against Fusarium solani and Fusarium kuroshium, causal agent of Fusarium dieback, by screening the rhizosphere and phyllosphere of three Lauraceae species. From 195 bacterial isolates, we identified 32 isolates that significantly reduced the growth of F. solani in vitro, which mostly belonged to bacterial taxa Bacillus, Pseudomonas and Actinobacteria. The antifungal activity of their volatile organic compounds (VOCs) was also evaluated. Bacterial strain Bacillus sp. CCeRi1-002, recovered from the rhizosphere of Aiouea effusa, showed the highest percentage of direct inhibition (62.5 %) of F. solani and produced diffusible compounds that significantly reduced its mycelial growth. HPLC-MS analyses on this strain allowed to tentatively identify bioactive compounds from three lipopeptide groups (iturin, surfactin and fengycin). Bacillus sp. CCeRi1-002 and another strain identified as Pseudomonas sp. significantly inhibited F. solani mycelial growth through the emission of VOCs. Chemical analysis of their volatile profiles indicated the likely presence of 2-nonanone, 2-undecanone, disulfide dimethyl and 1-butanol 3-methyl-, which had been previously reported with antifungal activity. In antagonism assays against F. kuroshium, Bacillus sp. CCeRi1-002 and its diffusible compounds exhibited significant antifungal activity and induced hyphal deformations. Our findings highlight the importance of considering bacteria associated with forest species and the need to include bacterial products in the search for potential antagonists of Fusarium dieback.

Microbiome-MX 2018: microbiota and microbiome opportunities in Mexico, a megadiverse country.

A weekly conference series paired with lectures entitled "Microbiome-MX: exploring the Microbiota and Microbiome Research in Mexico" was organized to provide a multidisciplinary overview of the most recent research done in Mexico using high-throughput sequencing. Scientists and postgraduate students from several disciplines such as microbiology, bioinformatics, virology, immunology, nutrition, and medical genomics gathered to discuss state of the art in each of their respective subjects of expertise, as well as advances, applications and new opportunities on microbiota/microbiome research. In particular, high-throughput sequencing is a crucial tool to understand the challenges of a megadiverse developing country as Mexico, and moreover to know the scientific capital and capabilities available for collaboration. The conference series addressed three main topics important for Mexico: i) the complex role of microbiota in health and prevalent diseases such as obesity, diabetes, inflammatory bowel disease, tuberculosis, HIV, autoimmune diseases and gastric cancer; ii) the use of local, traditional and prehispanic products as pre/probiotics to modulate the microbiota and improve human health; and iii) the impact of the microbiota in shaping the biodiversity of economically important terrestrial and marine ecosystems. Herein, we summarize the contributions that Mexican microbiota/microbiome research is making to the global trends, describing the highlights of the conferences and lectures, rather than a review of the state-of-the-art of this research. This meeting report also presents the efforts of a multidisciplinary group of scientist to encourage collaborations and bringing this research field closer for younger generations.

Smells from the desert: Microbial volatiles that affect plant growth and development of native and non-native plant species.

The plant microbiota can affect host fitness via the emission of microbial volatile organic compounds (mVOCs) that influence growth and development. However, evidence of these molecules and their effects in plants from arid ecosystems is limited. We screened the mVOCs produced by 40 core and representative members of the microbiome of agaves and cacti in their interaction with Arabidopsis thaliana and Nicotiana benthamiana. We used SPME-GC-MS to characterize the chemical diversity of mVOCs and tested the effects of selected compounds on growth and development of model and host plants. Our study revealed that approximately 90% of the bacterial strains promoted plant growth both in A. thaliana and N. benthamiana. Bacterial VOCs were mainly composed of esters, alcohols, and S-containing compounds with 25% of them not previously characterized. Remarkably, ethyl isovalerate, isoamyl acetate, 3-methyl-1-butanol, benzyl alcohol, 2-phenylethyl alcohol, and 3-(methylthio)-1-propanol, and some of their mixtures, displayed beneficial effects in A. thaliana and also improved growth and development of Agave tequilana and Agave salmiana in just 60 days. Volatiles produced by bacteria isolated from agaves and cacti are promising molecules for the sustainable production of crops in arid and semi-arid regions.

Functional Signatures of the Epiphytic Prokaryotic Microbiome of Agaves and Cacti.

Microbial symbionts account for survival, development, fitness and evolution of eukaryotic hosts. These microorganisms together with their host form a biological unit known as holobiont. Recent studies have revealed that the holobiont of agaves and cacti comprises a diverse and structured microbiome, which might be important for its adaptation to drylands. Here, we investigated the functional signatures of the prokaryotic communities of the soil and the episphere, that includes the rhizosphere and phyllosphere, associated with the cultivated Agave tequilana and the native and sympatric Agave salmiana, Opuntia robusta and Myrtillocactus geometrizans by mining shotgun metagenomic data. Consistent with previous phylogenetic profiling, we found that Proteobacteria, Actinobacteria and Firmicutes were the main represented phyla in the episphere of agaves and cacti, and that clustering of metagenomes correlated with the plant compartment. In native plants, genes related to aerobic anoxygenic phototrophy and photosynthesis were enriched in the phyllosphere and soil, while genes coding for biofilm formation and quorum sensing were enriched in both epiphytic communities. In the episphere of cultivated A. tequilana fewer genes were identified, but they belonged to similar pathways than those found in native plants. A. tequilana showed a depletion in several genes belonging to carbon metabolism, secondary metabolite biosynthesis and xenobiotic degradation suggesting that its lower microbial diversity might be linked to functional losses. However, this species also showed an enrichment in biofilm and quorum sensing in the epiphytic compartments, and evidence for nitrogen fixation in the rhizosphere. Aerobic anoxygenic phototrophic markers were represented by Rhizobiales (Methylobacterium) and Rhodospirillales (Belnapia) in the phyllosphere, while photosystem genes were widespread in Bacillales and Cyanobacteria. Nitrogen fixation and biofilm formation genes were mostly related to Proteobacteria. These analyses support the idea of niche differentiation in the rhizosphere and phyllosphere of agaves and cacti and shed light on the potential mechanisms by which epiphytic microbial communities survive and colonize plants of arid and semiarid ecosystems. This study establishes a guideline for testing the relevance of the identified functional traits on the microbial community and the plant fitness.

The Cacti Microbiome: Interplay between Habitat-Filtering and Host-Specificity.

Cactaceae represents one of the most species-rich families of succulent plants native to arid and semi-arid ecosystems, yet the associations Cacti establish with microorganisms and the rules governing microbial community assembly remain poorly understood. We analyzed the composition, diversity, and factors influencing above- and below-ground bacterial, archaeal, and fungal communities associated with two native and sympatric Cacti species: Myrtillocactus geometrizans and Opuntia robusta. Phylogenetic profiling showed that the composition and assembly of microbial communities associated with Cacti were primarily influenced by the plant compartment; plant species, site, and season played only a minor role. Remarkably, bacterial, and archaeal diversity was higher in the phyllosphere than in the rhizosphere of Cacti, while the opposite was true for fungi. Semi-arid soils exhibited the highest levels of microbial diversity whereas the stem endosphere the lowest. Despite their taxonomic distance, M. geometrizans and O. robusta shared most microbial taxa in all analyzed compartments. Influence of the plant host did only play a larger role in the fungal communities of the stem endosphere. These results suggest that fungi establish specific interactions with their host plant inside the stem, whereas microbial communities in the other plant compartments may play similar functional roles in these two species. Biochemical and molecular characterization of seed-borne bacteria of Cacti supports the idea that these microbial symbionts may be vertically inherited and could promote plant growth and drought tolerance for the fitness of the Cacti holobiont. We envision this knowledge will help improve and sustain agriculture in arid and semi-arid regions of the world.

Plant compartment and biogeography affect microbiome composition in cultivated and native Agave species.

Desert plants are hypothesized to survive the environmental stress inherent to these regions in part thanks to symbioses with microorganisms, and yet these microbial species, the communities they form, and the forces that influence them are poorly understood. Here we report the first comprehensive investigation of the microbial communities associated with species of Agave, which are native to semiarid and arid regions of Central and North America and are emerging as biofuel feedstocks. We examined prokaryotic and fungal communities in the rhizosphere, phyllosphere, leaf and root endosphere, as well as proximal and distal soil samples from cultivated and native agaves, through Illumina amplicon sequencing. Phylogenetic profiling revealed that the composition of prokaryotic communities was primarily determined by the plant compartment, whereas the composition of fungal communities was mainly influenced by the biogeography of the host species. Cultivated A. tequilana exhibited lower levels of prokaryotic diversity compared with native agaves, although no differences in microbial diversity were found in the endosphere. Agaves shared core prokaryotic and fungal taxa known to promote plant growth and confer tolerance to abiotic stress, which suggests common principles underpinning Agave-microbe interactions.

Diazotrophic potential among bacterial communities associated with wild and cultivated Agave species.

Agaves are major biotic resources in arid and semi-arid ecosystems. Despite their ecological, economical and cultural relevance, many aspects of the microbial communities associated with agaves are still unknown. Here, we investigated the bacterial communities associated with two Agave species by 16S rRNA- Denaturing gradient gel electrophoresis fingerprinting and sequencing. We also evaluated the effects of biotic and abiotic factors in the structure of the bacterial communities. In parallel, we isolated and characterized diazotrophic bacteria associated with agaves, as Agave soils are characterized by their low nitrogen content. Our results demonstrate that in Agave, the structure of prokaryotic assemblages was mostly influenced by the community group, where the soil, episphere, and endosphere were clearly distinct. Proteobacteria (γ and α), Actinobacteria, and Acidobacteria were the dominant phyla. Bacterial communities in the episphere of agaves were mainly influenced by the host species, whereas in the endosphere were affected by the season. Fifteen bacterial taxa were common and abundant in the endosphere of both Agave species during the dry season. Notably, some of the confirmed diazotrophic strains belonged to this group, suggesting a possible beneficial role in planta.

Evolution of small prokaryotic genomes.

As revealed by genome sequencing, the biology of prokaryotes with reduced genomes is strikingly diverse. These include free-living prokaryotes with ∼800 genes as well as endosymbiotic bacteria with as few as ∼140 genes. Comparative genomics is revealing the evolutionary mechanisms that led to these small genomes. In the case of free-living prokaryotes, natural selection directly favored genome reduction, while in the case of endosymbiotic prokaryotes neutral processes played a more prominent role. However, new experimental data suggest that selective processes may be at operation as well for endosymbiotic prokaryotes at least during the first stages of genome reduction. Endosymbiotic prokaryotes have evolved diverse strategies for living with reduced gene sets inside a host-defined medium. These include utilization of host-encoded functions (some of them coded by genes acquired by gene transfer from the endosymbiont and/or other bacteria); metabolic complementation between co-symbionts; and forming consortiums with other bacteria within the host. Recent genome sequencing projects of intracellular mutualistic bacteria showed that previously believed universal evolutionary trends like reduced G+C content and conservation of genome synteny are not always present in highly reduced genomes. Finally, the simplified molecular machinery of some of these organisms with small genomes may be used to aid in the design of artificial minimal cells. Here we review recent genomic discoveries of the biology of prokaryotes endowed with small gene sets and discuss the evolutionary mechanisms that have been proposed to explain their peculiar nature.

Evolution of an endofungal lifestyle: Deductions from the Burkholderia rhizoxinica genome.

BACKGROUND: Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic zygomycete Rhizopus microsporus, the causative agent of rice seedling blight. The endosymbiont produces the antimitotic macrolide rhizoxin for its host. It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. To shed light on the evolution and genetic potential of this model organism, we analysed the whole genome of B. rhizoxinica HKI 0454 - a type strain of endofungal Burkholderia species. RESULTS: The genome consists of a structurally conserved chromosome and two plasmids. Compared to free-living Burkholderia species, the genome is smaller in size and harbors less transcriptional regulator genes. Instead, we observed accumulation of transposons over the genome. Prediction of primary metabolic pathways and transporters suggests that endosymbionts consume host metabolites like citrate, but might deliver some amino acids and cofactors to the host. The rhizoxin biosynthesis gene cluster shows evolutionary traces of horizontal gene transfer. Furthermore, we analysed gene clusters coding for nonribosomal peptide synthetases (NRPS). Notably, B. rhizoxinica lacks common genes which are dedicated to quorum sensing systems, but is equipped with a large number of virulence-related factors and putative type III effectors. CONCLUSIONS: B. rhizoxinica is the first endofungal bacterium, whose genome has been sequenced. Here, we present models of evolution, metabolism and tools for host-symbiont interaction of the endofungal bacterium deduced from whole genome analyses. Genome size and structure suggest that B. rhizoxinica is in an early phase of adaptation to the intracellular lifestyle (genome in transition). By analysis of tranporters and metabolic pathways we predict how metabolites might be exchanged between the symbiont and its host. Gene clusters for biosynthesis of secondary metabolites represent novel targets for genomic mining of cryptic natural products. In silico analyses of virulence-associated genes, secreted proteins and effectors might inspire future studies on molecular mechanisms underlying bacterial-fungal interaction.

The microbe-free plant: fact or artifact?

Plant-microbe interactions are ubiquitous. Plants are threatened by pathogens, but they are even more commonly engaged in neutral or mutualistic interactions with microbes: belowground microbial plant associates are mycorrhizal fungi, Rhizobia, and plant-growth promoting rhizosphere bacteria, aboveground plant parts are colonized by internally living bacteria and fungi (endophytes) and by microbes in the phyllosphere (epiphytes). We emphasize here that a completely microbe-free plant is an exotic exception rather than the biologically relevant rule. The complex interplay of such microbial communities with the host-plant affects multiple vital parameters such as plant nutrition, growth rate, resistance to biotic and abiotic stressors, and plant survival and distribution. The mechanisms involved reach from direct ones such as nutrient acquisition, the production of plant hormones, or direct antibiosis, to indirect ones that are mediated by effects on host resistance genes or via interactions at higher trophic levels. Plant-associated microbes are heterotrophic and cause costs to their host plant, whereas the benefits depend on the current environment. Thus, the outcome of the interaction for the plant host is highly context dependent. We argue that considering the microbe-free plant as the "normal" or control stage significantly impairs research into important phenomena such as (1) phenotypic and epigenetic plasticity, (2) the "normal" ecological outcome of a given interaction, and (3) the evolution of plants. For the future, we suggest cultivation-independent screening methods using direct PCR from plant tissue of more than one fungal and bacterial gene to collect data on the true microbial diversity in wild plants. The patterns found could be correlated to host species and environmental conditions, in order to formulate testable hypotheses on the biological roles of plant endophytes in nature. Experimental approaches should compare different host-endophyte combinations under various relevant environmental conditions and study at the genetic, epigenetic, transcriptional, and physiological level the parameters that cause the interaction to shift along the mutualism-parasitism continuum.

Toxin production by bacterial endosymbionts of a Rhizopus microsporus strain used for tempe/sufu processing.

Mould fungi are not only well known for food spoilage through toxin formation but also for the production of fermented foods. In Asian countries, the fermentation of soy beans and tofu for tempe and sufu production with various Rhizopus strains is widespread. Here we report the finding of toxinogenic bacteria in a starter culture used for sufu production. By means of metabolic profiling of the fungus under standard conditions for tempe and sufu production, we found that toxins of the rhizoxin complex are produced in critical amounts. Considering that rhizoxins are severe toxins with strong antimitotic activity it is important to notice that our findings uncover a health-threatening symbiosis in food processing. A simple PCR method for detecting toxinogenic endofungal bacteria in starter cultures is proposed.

Endofungal bacteria as producers of mycotoxins.

Mycotoxins are compounds of fungal origin that can adversely affect human, animal and plant health through food spoilage or infection, even to the point of epidemics such as turkey X disease and ergotism. The biosynthetic pathways of various mycotoxins (such as aflatoxin and fumonisins) are generally well understood. However, two examples have recently been described where a mycotoxin is not synthesized by the fungus itself but by bacteria residing within the fungal cytosol. These discoveries have implications in various fields, such as ecology, medicine and food processing.