Ubiquitous arbuscular mycorrhizal fungi in the roots of herbaceous understory plants with hyphal degeneration in Colchicaceae and Gentianaceae.

Due to the loss of photosynthetic ability during evolution, some plant species rely on mycorrhizal fungi for their carbon source, and this nutritional strategy is known as mycoheterotrophy. Mycoheterotrophic plants forming Paris-type arbuscular mycorrhizas (AM) exhibit two distinctive mycorrhizal features: degeneration of fungal materials and specialization towards particular fungal lineages. To explore the possibility that some understory AM plants show partial mycoheterotrophy, i.e., both photosynthetic and mycoheterotrophic nutritional strategies, we investigated 13 green herbaceous plant species collected from five Japanese temperate forests. Following microscopic observation, degenerated hyphal coils were observed in four species: two Colchicaceae species, Disporum sessile and Disporum smilacinum, and two Gentianaceae species, Gentiana scabra and Swertia japonica. Through amplicon sequencing, however, we found that all examined plant species exhibited no specificity toward AM fungi. Several AM fungi were consistently found across most sites and all plant species studied. Because previous studies reported the detection of these AM fungi from various tree species in Japanese temperate forests, our findings suggest the presence of ubiquitous AM fungi in forest ecosystems. If the understory plants showing fungal degeneration exhibit partial mycoheterotrophy, they may obtain carbon compounds indirectly from a wide range of surrounding plants utilizing such ubiquitous AM fungi.

Distinct biogeographic patterns in Glomeromycotinian and Mucoromycotinian arbuscular mycorrhizal fungi across China: A meta-analysis.

Fine root endophytes, recently reclassified as Mucoromycotinian arbuscular mycorrhizal fungi (M-AMF), are now recognized as functionally important as Glomeromycotinian AMF (G-AMF). However, little is known about the biogeography and ecology of M-AMF and G-AMF communities, particularly on a large scale, preventing a systematic assessment of ecosystem diversity and functioning. Here, we investigated the biogeographic assemblies and ecological diversity patterns of both G-AMF and M-AMF, using published 18S rDNA amplicon datasets and associated metadata from 575 soil samples in six ecosystems across China. Contrasting with G-AMF, putative M-AMF were rare in natural/semi-natural sites, where their communities were a subset of those in agricultural sites characterized by intensive disturbances, suggesting different ecological niches that they could occupy. Spatial and environmental factors (e.g., vegetation type) significantly influenced both fungal communities, with soil total­nitrogen and mean-annual-precipitation being the strongest predictors for G-AMF and M-AMF richness, respectively. Both groups exhibited a strong spatial distance-decay relationship, shaped more by environmental filtering than spatial effects for M-AMF, and the opposite for G-AMF, presumably because stochasticity (e.g., drift) dominantly structured G-AMF communities; while the narrower niche breadth (at community-level) of M-AMF compared to G-AMF suggested its more susceptibility to environmental differences. Furthermore, co-occurrence network links between G-AMF and M-AMF were prevalent across ecosystems, and were predicted to play a key role in stabilizing overall communities harboring both fungi. Based on the macroecological spatial scale datasets, this study provides solid evidence that the two AMF groups have distinct ecological preferences at the continental scale in China, and also highlights the potential impacts of anthropogenic activities on distributions of AMF. These results advance our knowledge of the ecological differences between the two fungal groups in terrestrial ecosystems, suggesting the need for further field-based investigation that may lead to a more sophisticated understanding of ecosystem function and sustainable management.

GlobalAMFungi: a global database of arbuscular mycorrhizal fungal occurrences from high-throughput sequencing metabarcoding studies.

Arbuscular mycorrhizal (AM) fungi are crucial mutualistic symbionts of the majority of plant species, with essential roles in plant nutrient uptake and stress mitigation. The importance of AM fungi in ecosystems contrasts with our limited understanding of the patterns of AM fungal biogeography and the environmental factors that drive those patterns. This article presents a release of a newly developed global AM fungal dataset (GlobalAMFungi database, https://globalamfungi.com) that aims to reduce this knowledge gap. It contains almost 50 million observations of Glomeromycotinian AM fungal amplicon DNA sequences across almost 8500 samples with geographical locations and additional metadata obtained from 100 original studies. The GlobalAMFungi database is built on sequencing data originating from AM fungal taxon barcoding regions in: i) the small subunit rRNA (SSU) gene; ii) the internal transcribed spacer 2 (ITS2) region; and iii) the large subunit rRNA (LSU) gene. The GlobalAMFungi database is an open source and open access initiative that compiles the most comprehensive atlas of AM fungal distribution. It is designed as a permanent effort that will be continuously updated by its creators and through the collaboration of the scientific community. This study also documented applicability of the dataset to better understand ecology of AM fungal taxa.

Exploring the mycobiome and arbuscular mycorrhizal fungi associated with the rizosphere of the genus Inga in the pristine Ecuadorian Amazon.

This study explored the composition of the mycobiome in the rhizosphere of Inga seedlings in two different but neighboring forest ecosystems in the undisturbed tropical Amazon rainforest at the Tiputini Biodiversity Station in Ecuador. In terra firme plots, which were situated higher up and therefore typically outside of the influence of river floods, and in várzea plots, the lower part of the forest located near the riverbanks and therefore seasonally flooded, tree seedlings of the genus Inga were randomly collected and measured, and the rhizosphere soils surrounding the root systems was collected. Members of the Fabaceae family and the genus Inga were highly abundant in both forest ecosystems. Inga sp. seedlings collected in terra firme showed a lower shoot to root ratio compared to seedlings that were collected in várzea, suggesting that Inga seedlings which germinated in várzea soils could invest more resources in vegetative growth with shorter roots. Results of the physical-chemical properties of soil samples indicated higher proportions of N, Mo, and V in terra firme soils, whereas várzea soils present higher concentrations of all other macro- and micronutrients, which confirmed the nutrient deposition effect of seasonal flooding by the nearby river. ITS metabarcoding was used to explore the mycobiome associated with roots of the genus Inga. Bioinformatic analysis was performed using Qiime 2 to calculate the alpha and beta diversity, species taxonomy and the differential abundance of fungi and arbuscular mycorrhizal fungi. The fungal community represented 75% of the total ITS ASVs, and although present in all samples, the subphylum Glomeromycotina represented 1.42% of all ITS ASVs with annotations to 13 distinct families, including Glomeraceae (72,23%), Gigasporaceae (0,57%), Acaulosporaceae (0,49%). AMF spores of these three AMF families were morphologically identified by microscopy. Results of this study indicate that AMF surround the rhizosphere of Inga seedlings in relatively low proportions compared to other fungal groups but present in both terra firme and várzea Neotropical ecosystems.

Arbuscular mycorrhizal fungi associated with the rhizosphere of an endemic terrestrial bromeliad and a grass in the Brazilian neotropical dry forest.

Arbuscular mycorrhizal fungi form symbiotic associations with 80-90% of all known plants, allowing the fungi to acquire plant-synthesized carbon, and confer an increased capacity for nutrient uptake by plants, improving tolerance to abiotic and biotic stresses. We aimed at characterizing the mycorrhizal community in the rhizosphere of Neoglaziovia variegata (so-called `caroa`) and Tripogonella spicata (so-called resurrection plant), using high-throughput sequencing of the partial 18S rRNA gene. Both plants are currently undergoing a bioprospecting program to find microbes with the potential of helping plants tolerate water stress. Sampling was carried out in the Caatinga biome, a neotropical dry forest, located in northeastern Brazil. Illumina MiSeq sequencing of 37 rhizosphere samples (19 for N. variegata and 18 for T. spicata) revealed a distinct mycorrhizal community between the studied plants. According to alpha diversity analyses, T. spicata showed the highest richness and diversity based on the Observed ASVs and the Shannon index, respectively. On the other hand, N. variegata showed higher modularity of the mycorrhizal network compared to T. spicata. The four most abundant genera found (higher than 10%) were Glomus, Gigaspora, Acaulospora, and Scutellospora, with Glomus being the most abundant in both plants. Nonetheless, Gigaspora, Diversispora, and Ambispora were found only in the rhizosphere of N. variegata, whilst Scutellospora, Paraglomus, and Archaeospora were exclusive to the rhizosphere of T. spicata. Therefore, the community of arbuscular mycorrhizal fungi of the rhizosphere of each plant encompasses a unique composition, structure and modularity, which can differentially assist them in the hostile environment.

Structurally distinct mitoviruses: are they an ancestral lineage of the Mitoviridae exclusive to arbuscular mycorrhizal fungi (Glomeromycotina)?

Mitoviruses in the family Mitoviridae are the mitochondria-replicating "naked RNA viruses" with genomes encoding only the replicase RNA-dependent RNA polymerase (RdRp) and prevalent across fungi, plants, and invertebrates. Arbuscular mycorrhizal fungi in the subphylum Glomeromycotina are obligate plant symbionts that deliver water and nutrients to the host. We discovered distinct mitoviruses in glomeromycotinian fungi, namely "large duamitovirus," encoding unusually large RdRp with a unique N-terminal motif that is endogenized in some host genomes. More than 400 viral sequences similar to the large duamitoviruses are present in metatranscriptome databases. They are globally distributed in soil ecosystems, consistent with the cosmopolitan distribution of glomeromycotinian fungi, and formed the most basal clade of the Mitoviridae in phylogenetic analysis. Given that glomeromycotinian fungi are the only confirmed hosts of these viruses, we propose the hypothesis that large duamitoviruses are the most ancestral lineage of the Mitoviridae that have been maintained exclusively in glomeromycotinian fungi.

Isolation and identification of an arbuscular mycorrhizal fungus specifically associated with mycoheterotrophic seedlings of Gentiana zollingeri (Gentianaceae).

Gentiana zollingeri (Gentianaceae) is an initial mycoheterotrophic plant that depends on a specific group of arbuscular mycorrhizal (AM) fungi for carbon source during underground growth after seed germination. In this study, a mycorrhizal fungus dominant in mycoheterotrophic seedlings of G. zollingeri was successfully isolated from a soil core collected from a point close to a flowering G. zollingeri. The AM fungal isolate was identified as conspecific or closely related to Dominikia aurea (Glomeraceae) by spore morphology and molecular phylogeny. Basic Local Alignment Search Tool (BLAST) searches against the MaarjAM database showed that the nuclear small subunit ribosomal DNA sequences of the isolate matched the AM fungal sequences obtained from a wide range of plants in various ecosystems, including several mycoheterotrophs. Thus, it is suggested that the AM fungal isolate is one of the cheating susceptible AM fungi. Furthermore, the sequences corresponded to those of a group of AM fungi dominantly detected in Japanese temperate forests. Accordingly, there is a possibility that mycoheterotrophic plants, including seedlings of G. zollingeri, may target AM fungi with a wide host range and ubiquitous distribution.

Generalist arbuscular mycorrhizal fungi dominated heavy metal polluted soils at two artisanal and small - scale gold mining sites in southeastern Ecuador.

BACKGROUND: Artisanal and small-scale gold mining activities are producing contamination with heavy metals and metalloids (HMM) into soils and water worldwide. The HMM are considered as one of the major abiotic stresses due to their long-term persistence in soil. In this context, arbuscular mycorrhizal fungi (AMF) confer resistance to a variety of abiotic plant stressors including HMM. However, little is known regarding the diversity and composition of AMF communities in heavy metal polluted sites in Ecuador. METHODS: In order to investigate the AMF diversity, root samples and associated soil of six plant species were collected from two sites polluted by heavy metals, located in Zamora-Chinchipe province, Ecuador. The AMF 18S nrDNA genetic region was analyzed and sequenced, and fungal OTUs were defined based on 99% sequence similarity. Results were contrasted with AMF communities from a natural forest and from reforestation sites located in the same province and with available sequences in GenBank. RESULTS: The main pollutants in soils were Pb, Zn, Hg, Cd and Cu with concentrations exceeding the soil reference value for agricultural use. Molecular phylogeny and OTU delimitation showed 19 OTUs, the family Glomeraceae was the most OTU-rich followed by Archaeosporaceae, Acaulosporaceae, Ambisporaceae and Paraglomeraceae. Most of the OTUs (11 of 19) have been found at other locations worldwide, 14 OTUs were proven from nearby non-contaminated sites in Zamora-Chinchipe. CONCLUSION: Our study showed that there are no specialized OTUs at the studied HMM polluted sites, but rather generalists adapted to a wide variety of habitats. Their potential role in phytoremediation approaches remains to be investigated.

Clonal spore populations in sporocarps of arbuscular mycorrhizal fungi.

Some arbuscular mycorrhizal (AM) fungal species known to form sporocarps (i.e., aggregations of spores) are polyphyletic in two orders, Glomerales and Diversisporales. Spore clusters (sporocarp-like structures) often formed in pot cultures or in vitro conditions are supposed to be clonal populations, while sporocarps in natural habitats with a fungal peridium are morphologically similar to those of epigeous sexual (zygosporic) sporocarps of Endogone species. Thus, in this study, we explored the genetics of sporocarpic spores of two AM fungi with a view to possibilities of clonal or sexual reproduction during sporocarps formation. To examine these possibilities, we investigated single-nucleotide polymorphisms (SNPs) in reduced genomic libraries of spores isolated from sporocarps molecularly identified as Rhizophagus irregularis and Diversispora epigaea. In addition, partial sequences of the MAT locus HD2 gene of R. irregularis were phylogenetically analyzed to determine the nuclear status of the spores. We found that most SNPs were shared among the spores isolated from each sporocarp in both species. Furthermore, all HD2 sequences from spores isolated from three R. irregularis sporocarps were identical. These results indicate that those sporocarps comprise clonal spores. Therefore, sporocarps with clonal spores may have different functions than sexual reproduction, such as massive spore production or spore dispersal via mycophagy.

A targeted bioinformatics approach identifies highly variable cell surface proteins that are unique to Glomeromycotina.

Diversity in arbuscular mycorrhizal fungi (AMF) contributes to biodiversity and resilience in natural environments and healthy agricultural systems. Functional complementarity exists among species of AMF in symbiosis with their plant hosts, but the molecular basis of this is not known. We hypothesise this is in part due to the difficulties that current sequence assembly methodologies have assembling sequences for intrinsically disordered proteins (IDPs) due to their low sequence complexity. IDPs are potential candidates for functional complementarity because they often exist as extended (non-globular) proteins providing additional amino acids for molecular interactions. Rhizophagus irregularis arabinogalactan-protein-like proteins (AGLs) are small secreted IDPs with no known orthologues in AMF or other fungi. We developed a targeted bioinformatics approach to identify highly variable AGLs/IDPs in RNA-sequence datasets. The approach includes a modified multiple k-mer assembly approach (Oases) to identify candidate sequences, followed by targeted sequence capture and assembly (mirabait-mira). All AMF species analysed, including the ancestral family Paraglomeraceae, have small families of proteins rich in disorder promoting amino acids such as proline and glycine, or glycine and asparagine. Glycine- and asparagine-rich proteins also were found in Geosiphon pyriformis (an obligate symbiont of a cyanobacterium), from the same subphylum (Glomeromycotina) as AMF. The sequence diversity of AGLs likely translates to functional diversity, based on predicted physical properties of tandem repeats (elastic, amyloid, or interchangeable) and their broad pI ranges. We envisage that AGLs/IDPs could contribute to functional complementarity in AMF through processes such as self-recognition, retention of nutrients, soil stability, and water movement.

Agricultural land-use favours Mucoromycotinian, but not Glomeromycotinian, arbuscular mycorrhizal fungi across ten biomes.

Globally, agricultural land-use negatively affects soil biota that contribute to ecosystem functions such as nutrient cycling, yet arbuscular mycorrhizal fungi (AMF) are promoted as essential components of agroecosystems. Arbuscular mycorrhizal fungi include Glomeromycotinian AMF (G-AMF) and the arbuscule-producing fine root endophytes, recently re-classified into the Endogonales order within Mucoromycotina. The correct classification of Mucoromycotinian AMF (M-AMF) and the availability of new molecular tools can guide research to better the understanding of their diversity and ecology. To investigate the impact on G-AMF and M-AMF of agricultural land-use at a continental scale, we sampled DNA from paired farm and native sites across 10 Australian biomes. Glomeromycotinian AMF were present in both native and farm sites in all biomes. Putative M-AMF were favoured by farm sites, rare or absent in native sites, and almost entirely absent in tropical biomes. Temperature, rainfall, and soil pH were strong drivers of richness and community composition of both groups, and plant richness was an important mediator. Both fungal groups occupy different, but overlapping, ecological niches, with M-AMF thriving in temperate agricultural landscapes. Our findings invite exploration of the origin and spread of M-AMF and continued efforts to resolve the phylogeny of this newly reclassified group of AMF.

Dispersal of Arbuscular Mycorrhizal Fungi: Evidence and Insights for Ecological Studies.

Dispersal is a critical ecological process that modulates gene flow and contributes to the maintenance of genetic and taxonomic diversity within ecosystems. Despite an increasing global understanding of the arbuscular mycorrhizal (AM) fungal diversity, distribution and prevalence in different biomes, we have largely ignored the main dispersal mechanisms of these organisms. To provide a geographical and scientific overview of the available data, we systematically searched for the direct evidence on the AM fungal dispersal agents (abiotic and biotic) and different propagule types (i.e. spores, extraradical hyphae or colonized root fragments). We show that the available data (37 articles) on AM fungal dispersal originates mostly from North America, from temperate ecosystems, from biotic dispersal agents (small mammals) and AM fungal spores as propagule type. Much lesser evidence exists from South American, Asian and African tropical systems and other dispersers such as large-bodied birds and mammals and non-spore propagule types. We did not find strong evidence that spore size varies across dispersal agents, but wind and large animals seem to be more efficient dispersers. However, the data is still too scarce to draw firm conclusions from this finding. We further discuss and propose critical research questions and potential approaches to advance the understanding of the ecology of AM fungi dispersal.

Evidence for Niche Differentiation in the Environmental Responses of Co-occurring Mucoromycotinian Fine Root Endophytes and Glomeromycotinian Arbuscular Mycorrhizal Fungi.

Fine root endophytes (FRE) were traditionally considered a morphotype of arbuscular mycorrhizal fungi (AMF), but recent genetic studies demonstrate that FRE belong within the subphylum Mucoromycotina, rather than in the subphylum Glomeromycotina with the AMF. These findings prompt enquiry into the fundamental ecology of FRE and AMF. We sampled FRE and AMF in roots of Trifolium subterraneum from 58 sites across temperate southern Australia. We investigated the environmental drivers of composition, richness, and root colonization of FRE and AMF by using structural equation modelling and canonical correspondence analyses. Root colonization by FRE increased with increasing temperature and rainfall but decreased with increasing phosphorus (P). Root colonization by AMF increased with increasing soil organic carbon but decreased with increasing P. Richness of FRE decreased with increasing temperature and soil pH. Richness of AMF increased with increasing temperature and rainfall but decreased with increasing soil aluminium (Al) and pH. Aluminium, soil pH, and rainfall were, in decreasing order, the strongest drivers of community composition of FRE; they were also important drivers of community composition of AMF, along with temperature, in decreasing order: rainfall, Al, temperature, and soil pH. Thus, FRE and AMF showed the same responses to some (e.g. soil P, soil pH) and different responses to other (e.g. temperature) key environmental factors. Overall, our data are evidence for niche differentiation among these co-occurring mycorrhizal associates.

Prevalence and phenology of fine root endophyte colonization across populations of Lycopodiella inundata.

Mycorrhizal fungi are critical components of terrestrial habitats and agroecosystems. Recently, Mucoromycotina fine root endophyte fungi (MucFRE) were found to engage in nutritional mutualism with Lycopodiella inundata, which belongs to one of the earliest vascular plant lineages known to associate with MucFRE. The extent to which this mutualism plays a role in resilient plant populations can only be understood by examining its occurrence rate and phenological patterns. To test for prevalence and seasonality in colonization, we examined 1305 individual L. inundata roots from 275 plants collected during spring and autumn 2019 across 11 semi-natural heathlands in Britain and the Netherlands. We quantified presence/absence of fine root endophyte (FRE) hyphae and vesicles and explored possible relationships between temperature and precipitation in the months immediately before sampling. Fine root endophyte hyphae were dominant in all of the examined heathlands, and every colonized root had FRE in both cortical cells and root hairs. However, we found significant differences in colonization between the two seasons at every site. Overall, 14% of L. inundata roots were colonized in spring (2.4% with vesicles) compared with 86% in autumn (7.6% with vesicles). Colonization levels between populations were also significantly different, correlating with temperature and precipitation, suggesting some local environments may be more conducive to root and related hyphal growth. These marked seasonal differences in host-plant colonization suggest that results about FRE from single time point collections should be carefully interpreted. Our findings are relevant to habitat restoration, species conservation plans, agricultural bio-inoculation treatments, and microbial diversity studies.

Community Structure of Arbuscular Mycorrhizal Fungi in Soils of Switchgrass Harvested for Bioenergy.

Learning more about the biodiversity and composition of arbuscular mycorrhizal fungi (AMF) under alternative agricultural management scenarios may be important to the sustainable intensification of switchgrass grown as a bioenergy crop. Using PacBio single-molecule sequencing and taxonomic resolution to the level of amplicon sequence variant (ASV), we assessed the effects of nitrogen amendment on AMF associating with switchgrass and explored relationships between AMF and switchgrass yield across three sites of various productivities in Wisconsin. Nitrogen amendment had little effect on AMF diversity metrics or community composition. While AMF ASV diversity was not correlated with switchgrass yield, AMF family richness and switchgrass yield had a strong, positive relationship at one of our three sites. Each of our sites was dominated by unique ASVs of the species Paraglomus brasilianum, indicating regional segregation of AMF at the intraspecific level. Our molecular biodiversity survey identified putative core members of the switchgrass microbiome, as well as novel clades of AMF, especially in the order Paraglomerales and the genus Nanoglomus Furthermore, our phylogenies unite the cosmopolitan, soil-inhabiting clade deemed GS24 with Pervetustaceae, an enigmatic family prevalent in stressful environments. Future studies should isolate and characterize the novel genetic diversity found in switchgrass agroecosystems and explore the potential yield benefits of AMF richness.IMPORTANCE We assessed the different species of beneficial fungi living in agricultural fields of switchgrass, a large grass grown for biofuels, using high-resolution DNA sequencing. Contrary to our expectations, the fungi were not greatly affected by fertilization. However, we found a positive relationship between plant productivity and the number of families of beneficial fungi at one site. Furthermore, we sequenced many species that could not be identified with existing reference databases. One group of fungi was highlighted in an earlier study for being widely distributed but of unknown taxonomy. We discovered that this group belonged to a family called Pervetustaceae, which may benefit switchgrass in stressful environments. To produce higher-yielding switchgrass in a more sustainable manner, it could help to study these undescribed fungi and the ways in which they may contribute to greater switchgrass yield in the absence of fertilization.

Communities of arbuscular mycorrhizal fungi in forest ecosystems in Japan's temperate region may be primarily constituted by limited fungal taxa.

We investigated arbuscular mycorrhizal (AM) fungal communities in secondary forests and/or Chamaecyparis obtusa plantations at eight study sites in Japan's temperate region. In the secondary forests, AM plants of the families Lauraceae, Sapindaceae, Rutaceae, Araliaceae, Rosaceae, Magnoliaceae, Cornaceae, Piperaceae, and Anacardiaceae were found. The AM fungal communities were evaluated based on compositions of the AM fungal operational taxonomic units (OTUs), which were clustered at a 97% similarity threshold of the sequences of a partial small subunit of a nuclear ribosomal RNA gene obtained from the plant roots. The compositions of AM fungal OTUs were significantly correlated with the plant family compositions and were significantly differentiated among the study sites and between the study forests. Interestingly, only 19 OTUs remained after selecting for those that had more than 1.0% of the total reads, and these 19 OTUs accounted for 86.3% of the total rarefied reads that were classified into 121 OTUs. Furthermore, three dominant OTUs constituted 48.0% of the total reads, and the most dominant OTU was found at all study sites, except at one. These results indicate that AM fungal communities are primarily constituted by limited AM fungal taxa in the forest ecosystems with diverse plant taxa in Japan's temperate region. The results of basic local algorithm search tool (BLAST) searches against MaarjAM, a database of AM fungal sequences, also revealed that the AM fungi which were the three dominant OTUs are distributed in forest ecosystems on a worldwide scale.

A low cost alternative, using mycorrhiza and organic fertilizer, to optimize the production of foliar bioactive compounds in pomegranates.

AIM: To select the best combination of arbuscular mycorrhizal fungi and efficient vermicompost dose in maximizing the production of leaf metabolites in Punica granatum seedlings. METHODS AND RESULTS: The experimental design was in a 3 × 3 factorial arrangement: three inoculation treatments (inoculated with Gigaspora albida, inoculated with Acaulospora longula and control not inoculated) × 3 doses of vermicompost (0, 5 and 7·5%). After 120 days of inoculation, biomolecules, plant growth parameters and mycorrhizal colonization were evaluated. The combination of 7·5% of vermicompost and A. longula was favourable to the accumulation of leaf phenols, with an increase of 116·11% in relation to the non-inoculated control. The total tannins was optimized/enhanced when G. albida and 7·5% of fertilizer were used, registering an increase of 276·71%. CONCLUSIONS: The application of 7·5% of vermicompost associated with A. longula and G. albida is a low cost alternative to increase the levels of bioactive compounds in pomegranate leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first published report of optimization of bioactive compound production in P. granatum by the combined use of mycorrhiza and vermicompost doses.