Cost-efficient production of in vitro Rhizophagus irregularis.

One of the bottlenecks in mycorrhiza research is that arbuscular mycorrhizal fungi (AMF) have to be cultivated with host plant roots. Some AMF species, such as Rhizophagus irregularis, can be grown in vitro on dual-compartment plates, where fungal material can be harvested from a fungus-only compartment. Plant roots often grow into this fungus compartment, and regular root trimming is required if the fungal material needs to be free of traces of plant material. Trimming also increases unwanted contamination by other microorganisms. We compared 22 different culture types and conditions to a widely used dual-compartment culture system that we refer to as the "standard system." We found two modified culture systems that allowed high spore production and low rates of contamination. We then compared the two modified culture systems with the standard system in more detail. In the two modified culture systems versus the standard system, a comparable number of spores were produced per plate, the necessity for root trimming was reduced, and there was significantly diminished contamination in the fungal compartment. A cost analysis showed that both modified culture systems were more economic than the standard culture system for the production of the same number of non-contaminated spores. The two modified culture systems provide an economic alternative for the production of contaminant-free fungal material which is ideal for studies requiring AMF DNA or RNA for genetics, genomics, and transcriptomic studies or for studies requiring relatively large amounts of fungal material for greenhouse experiments.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Investigating unexplained genetic variation and its expression in the arbuscular mycorrhizal fungus Rhizophagus irregularis: A comparison of whole genome and RAD sequencing data.

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants. Recently, studies of the AMF Rhizophagus irregularis recorded within-isolate genetic variation that does not completely match the proposed homokaryon or heterokaryon state (where heterokaryons comprise a population of two distinct nucleus genotypes). We re-analysed published data showing that bi-allelic sites (and their frequencies), detected in proposed homo- and heterokaryote R. irregularis isolates, were similar across independent studies using different techniques. This indicated that observed within-fungus genetic variation was not an artefact of sequencing and that such within- fungus genetic variation possibly exists. We then looked to see if bi-allelic transcripts from three R. irregularis isolates matched those observed in the genome as this would give a strong indication of whether bi-allelic sites recorded in the genome were reliable variants. In putative homokaryon isolates, very few bi-allelic transcripts matched those in the genome. In a putative heterokaryon, a large number of bi-allelic transcripts matched those in the genome. Bi-allelic transcripts also occurred in the same frequency in the putative heterokaryon as predicted from allele frequency in the genome. Our results indicate that while within-fungus genome variation in putative homokaryon and heterokaryon AMF was highly similar in 2 independent studies, there was little support that this variation is transcribed in homokaryons. In contrast, within-fungus variation thought to be segregated among two nucleus genotypes in a heterokaryon isolate was indeed transcribed in a way that is proportional to that seen in the genome.

Population genomics reveals that within-fungus polymorphism is common and maintained in populations of the mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal (AM) fungi are symbionts of most plants, increasing plant growth and diversity. The model AM fungus Rhizophagus irregularis (isolate DAOM 197198) exhibits low within-fungus polymorphism. In contrast, another study reported high within-fungus variability. Experiments with other R. irregularis isolates suggest that within-fungus genetic variation can affect the fungal phenotype and plant growth, highlighting the biological importance of such variation. We investigated whether there is evidence of differing levels of within-fungus polymorphism in an R. irregularis population. We genotyped 20 isolates using restriction site-associated DNA sequencing and developed novel approaches for characterizing polymorphism among haploid nuclei. All isolates exhibited higher within-isolate poly-allelic single-nucleotide polymorphism (SNP) densities than DAOM 197198 in repeated and non-repeated sites mapped to the reference genome. Poly-allelic SNPs were independently confirmed. Allele frequencies within isolates deviated from diploids or tetraploids, or that expected for a strict dikaryote. Phylogeny based on poly-allelic sites was robust and mirrored the standard phylogeny. This indicates that within-fungus genetic variation is maintained in AM fungal populations. Our results predict a heterokaryotic state in the population, considerable differences in copy number variation among isolates and divergence among the copies, or aneuploidy in some isolates. The variation may be a combination of all of these hypotheses. Within-isolate genetic variation in R. irregularis leads to large differences in plant growth. Therefore, characterizing genomic variation within AM fungal populations is of major ecological importance.