Blots were performed against DDR (p53 pSer15, histone 2AX pSer139), cell survival/ cell death (AKT pThr308, cleaved PARP), and cell signaling (ERK1/2 pThr202/Tyr204) markers and controls. Actin and GAPDH served as loading controls.

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.

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.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00572-017-0763-2) contains supplementary material, which is available to authorized users.

Tel: +41 79 536 7546 12 13 14 2 The unprecedented challenge to feed the rapidly growing human population can only be 15 achieved with major changes in how we combine technology with agronomy 1 . Despite their 16 potential few beneficial microbes have truly been demonstrated to significantly increase 17 productivity of globally important crops in real farming conditions 2,3 . The way microbes are 18 employed has largely ignored the successes of crop breeding where naturally occurring 19 intraspecific variation of plants has been used to increase yields. Doing this with microbes 20 requires establishing a link between variation in the microbes and quantitative traits of crop 21 growth along with a clear demonstration that intraspecific microbial variation can potentially 22 lead to large differences in crop productivity in real farming conditions. Arbuscular mycorrhizal 23 fungi (AMF), form symbioses with globally important crops and show great potential to improve 24 crop yields 2 . Here we demonstrate the first link between patterns of genome-wide intraspecific 25 AMF variation and productivity of the globally important food crop cassava. Cassava, one of the 26 most important food security crops, feeds approximately 800 million people daily 4 . In 27 subsequent field trials, inoculation with genetically different isolates of the AMF Rhizophagus 28 irregularis altered cassava root productivity by up to 1.46-fold in conventional cultivation in 29 Colombia. In independent field trials in Colombia, Kenya and Tanzania, clonal sibling progeny 30 of homokaryon and dikaryon parental AMF enormously altered cassava root productivity by up 31 to 3 kg per plant and up to a 3.69-fold productivity difference. Siblings were clonal and, thus, 32 qualitatively genetically identical. Heterokaryon siblings can vary quantitatively but monokaryon 33 siblings are identical. Very large among-AMF sibling effects were observed at each location 34 although which sibling AMF was most effective depended strongly on location and cassava 35 variety. We demonstrate the enormous potential of genetic, and possibly epigenetic variation, in 36 AMF to greatly alter productivity of a globally important crop that should not be ignored. A 37 microbial improvement program to accelerate crop yield increases over that possible by plant 38 breeding or GMO technology alone is feasible. However, such a paradigm shift can only be 39 realised if researchers address how plant genetics and local environments affect mycorrhizal 40 responsiveness of crops to predict which fungal variant will be effective in a given location.41 For millennia farmers have improved crops using naturally occurring intraspecific plant genetic variation 42 to improve productivity. However, rates of yield increase attributed to plant breeding and GMO-crop 43 technology are not considered sufficient to feed the projected global human population 1 . Beneficial soil 65 there was a significant phylogenetic signal on spore density and clustering (Supplementary figure 1; 66Supplementary infor...