Exogenous abscisic acid and root volatiles increase sporulation of Rhizophagus irregularis DAOM 197198 in asymbiotic and pre-symbiotic status Abstract Several studies have demonstrated asymbiotic growth and development of arbuscular mycorrhizal (AM) fungi, although AM fungi are regarded as obligately symbiotic root-inhabiting fungi. Phytohormones, root exudates, and volatiles are important factors regulating the host-AM fungi interaction. However, the effects of phytohormones, root exudates, and volatiles on asymbiotic (without roots present) or pre-symbiotic (with roots present but no colonization) sporulation of AM fungi are unexplored. In this study, we tested the asymbiotic sporulation of Rhizophagus irregularis DAOM 197198 and further investigated the influences of abscisic acid (ABA), the exudates, and volatiles of tomato hairy roots on asymbiotic or pre-symbiotic sporulation in vitro. Results indicated that mother spores asymbiotically and pre-symbiotically produced daughter spores singly or in pairs. Compared with symbiotically produced spores, pre-symbiotically produced spores were significantly smaller (43.1 μm vs. 89.2 μm in diameter). Exogenous ABA applied to mother spores significantly increased the number of daughter spores, and root volatiles also significantly promoted pre-symbiotic sporulation. Our results provide the first evidence that exogenous ABA can promote AM fungal asymbiotic and pre-symbiotic sporulation, which highlights the potential role of phytohormones in AM fungal propagation.

In vitro mycorrhization of pear ( Pyrus communis ) Abstract The Mycelium Donor Plant system (MDP) was adapted to study the time course of the colonization of Pyrus communis by Rhizophagus irregularis under in vitro conditions. Isolated germinated spores did not colonize pear roots. Inoculum composed of R. irregularis spores/mycelium associated with chicory root fragments was used to inoculate Medicago truncatula which became thereafter the MDP of pear plantlets. Typical intraradical structures (hyphae, arbuscules, spores/vesicles) and appressoria were observed in the pear roots. During acclimatization, the pear plants formed a densely branched root system. R. irregularis colonization not only altered the root architecture but also changed the nutrient composition of the acclimatized pear plantlets.

Geography and habitat predominate over climate influences on arbuscular mycorrhizal fungal communities of mid-European meadows Abstract Despite the crucial importance of arbuscular mycorrhizal fungi (AMF) for numerous processes within terrestrial ecosystems, knowledge of the determinants of AMF community structure still is limited, mainly because of the limited scope of the available individual case studies which often only include a few environmental variables. Here, we describe the AMF diversity of mid-European meadows (mown or regularly cut grasslands, or recently abandoned lands where grasslands established spontaneously) within a considerably heterogeneous landscape over a scale of several hundred kilometers with regard to macroclimatic, microclimatic, and soil parameters. We include data describing the habitat (including vegetation type), geography, and climate, and test their contribution to the structure of the AMF communities at a regional scale. We amplified and sequenced the ITS 2 region of the ribosomal DNA operon of the AMF from soil samples using nested PCR and Illumina pair-end amplicon sequencing. Habitat (especially soil pH) and geographical parameters (spatial distance, altitude, and longitude) were the main determinants of the structure of the AMF communities in the meadows at a regional scale, with the abundance of genera Septoglomus, Paraglomus, Archaeospora, Funneliformis, and Dominikia driving the main response. The effects of climate and vegetation type were not significant and were mainly encompassed within the geography and/or soil pH effects. This study illustrates how important it is to have a large set of environmental metadata to compare the importance of different factors influencing the AMF community structure at large spatial scales.

Soil spore bank in Tuber melanosporum : up to 42% of fruitbodies remain unremoved in managed truffle grounds Abstract Fungi fruiting hypogeously are believed to form spore banks in soil especially because some fruitbodies are not removed by animals. However, little is known on the proportion of fruitbodies that are not removed by animals. We took advantage of the brûlé phenomenon, which allows delineation of the mycelium distribution, to assess the proportion of unremoved black truffle (Tuber melanosporum) fruitbodies in the context of plantations where fruitbodies are actively sought and harvested by truffle growers. We inspected portions of the brûlés after the harvest season to find unremoved fruitbodies. On average, from six truffle grounds in which a total of 38 brûlés were investigated, unremoved fruitbodies represented 33% of the whole fruitbody production (42% when averaging all the brûlés). We discuss this value and its high variability among truffle grounds. Beyond the local and variable accidental reasons that may lead to this high proportion, we speculate that the formation of some undetectable fruitbodies may be under selection pressure, given the reproductive biology of T. melanosporum.

How deep can ectomycorrhizas go? A case study on Pisolithus down to 4 meters in a Brazilian eucalypt plantation Abstract Despite the strong ecological importance of ectomycorrhizal (ECM) fungi, their vertical distribution remains poorly understood. To our knowledge, ECM structures associated with trees have never been reported in depths below 2 meters. In this study, fine roots and ECM root tips were sampled down to 4-m depth during the digging of two independent pits differing by their water availability. A meta-barcoding approach based on Illumina sequencing of internal transcribed spacers (ITS1 and ITS2) was carried out on DNA extracted from root samples (fine roots and ECM root tips separately). ECM fungi dominated the root-associated fungal community, with more than 90% of sequences assigned to the genus Pisolithus. The morphological and barcoding results demonstrated, for the first time, the presence of ECM symbiosis down to 4-m. The molecular diversity of Pisolithus spp. was strongly dependent on depth, with soil pH and soil water content as primary drivers of the Pisolithus spp. structure. Altogether, our results highlight the importance to consider the ECM symbiosis in deep soil layers to improve our understanding of fine roots functioning in tropical soils.

Axenic growth of the arbuscular mycorrhizal fungus Rhizophagus irregularis and growth stimulation by coculture with plant growth-promoting rhizobacteria Abstract Arbuscular mycorrhizal (AM) fungi are ecologically important for the growth and survival of most vascular plants. These fungi are known as obligate biotrophs that acquire carbon solely from host plants. A 13C-labeling experiment revealed the ability of axenically grown Rhizophagus irregularis DAOM 197198 to derive carbon from axenic culture on a relatively novel medium containing two sources of palmitic acid developed by Ishii (designated IH medium). In a separate experiment, this model fungus grew larger mycelia and produced more daughter spores on the IH medium in the presence of two Variovorax paradoxus strains than in axenic culture. In contrast, a strain of Mycobacterium sp. did not influence the growth of the AM fungus. Rhizophagus irregularis produced branched absorbing structures on the IH medium and, in monoxenic culture with V. paradoxus, sometimes formed densely packed hyphal coils. In this study, we report for the first time the formation of coarse terminal pelotons and of terminal and intercalary very fine (≈ 1 μm diameter) hyphal elongations, which could form daughter spores in the presence of V. paradoxus. This study shows the value of IH medium and certain rhizobacteria in the culture of R. irregularis DAOM 197198 in vitro.

Rhizophagus intraradices promotes alfalfa ( Medicago sativa ) defense against pea aphids ( Acyrthosiphon pisum ) revealed by RNA-Seq analysis Abstract Pea aphids (Acyrthosiphon pisum) are one of the most important insect pests of alfalfa (Medicago sativa). Arbuscular mycorrhizal (AM) fungi are important microorganisms of the agroecosystem that promote plant growth and improve plant resistance to abiotic and biotic stress. Little information is available on AM fungi-regulated defense responses of alfalfa to pea aphids. To better understand how alfalfa responds and to evaluate the impact of an AM fungus on aphid infestation, transcriptome sequencing was done and physiological parameters were analyzed. Our experiments showed that Rhizophagus intraradices can regulate plant response to aphids by promoting growth and increasing plant peroxidase (POD) and catalase (CAT) activities and salicylic acid (SA) concentration after aphid infestation. Transcriptome analysis showed that R. intraradices increased the expression of resistance-related genes, such as “WRKY transcription factor” and “Kunitz trypsin inhibitor.” Additionally, GO terms “chitinase activity,” “peroxidase activity,” “defense response,” and “response to biotic stimulus,” and KEGG pathways “phenylpropanoid biosynthesis” and “phenylalanine metabolism” were significantly enriched in mycorrhizal fungus-inoculated plants and aphid-infested plants. These findings will improve our understanding about the impact of this AM fungus on alfalfa response to aphid feeding and will provide the basis for further research on plant defense against aphids.

Evolution and networks in ancient and widespread symbioses between Mucoromycotina and liverworts Abstract Like the majority of land plants, liverworts regularly form intimate symbioses with arbuscular mycorrhizal fungi (Glomeromycotina). Recent phylogenetic and physiological studies report that they also form intimate symbioses with Mucoromycotina fungi and that some of these, like those involving Glomeromycotina, represent nutritional mutualisms. To compare these symbioses, we carried out a global analysis of Mucoromycotina fungi in liverworts and other plants using species delimitation, ancestral reconstruction, and network analyses. We found that Mucoromycotina are more common and diverse symbionts of liverworts than previously thought, globally distributed, ancestral, and often co-occur with Glomeromycotina within plants. However, our results also suggest that the associations formed by Mucoromycotina fungi are fundamentally different because, unlike Glomeromycotina, they may have evolved multiple times and their symbiotic networks are un-nested (i.e., not forming nested subsets of species). We infer that the global Mucoromycotina symbiosis is evolutionarily and ecologically distinctive.

Increased maize growth and P uptake promoted by arbuscular mycorrhizal fungi coincide with higher foliar herbivory and larval biomass of the Fall Armyworm Spodoptera frugiperda Abstract Most plant species naturally associate with arbuscular mycorrhizal fungi (AMF), which are known to promote crop nutrition and health in agroecosystems. However, information on how mycorrhizal associations affect plant biotic interactions that occur aboveground with foliar herbivores is limited and needs to be further addressed for the development of pest management strategies. With the objective to examine the influence of maize mycorrhizas on foliar herbivory caused by larvae of Spodoptera frugiperda, a serious pest in maize agroecosystems, we performed a fully factorial greenhouse pot experiment with three factors: Maize genotype (Puma and Milpal H318), AMF (with and without AMF, and without AMF with mineral P) and Insect herbivory (with and without S. frugiperda). Main results showed that inoculation with AMF improved plant growth and foliar P concentration, which coincided with increased foliar damage from herbivory and higher biomass of S. frugiperda larvae. A significant positive correlation between shoot P concentration and larval biomass was also observed. Finally, foliar herbivory by S. frugiperda slightly increased and decreased AMF root colonization in Puma and H318, respectively. In conclusion, our results show that maize plant benefits from AMF in terms of promotion of growth and nutrition, and may also increase the damage caused from insects by improving the food quality of maize leaves for larval growth, which seems to be linked to increased P uptake by the maize mycorrhizal association.

The European delicacy Tuber melanosporum forms mycorrhizae with some indigenous Chinese Quercus species and promotes growth of the oak seedlings Abstract We aimed to test whether Tuber melanosporum and native Chinese oak species could form stable mycorrhizal symbioses. Six oak species were all either inoculated or not, with spores of the Périgord black truffle in the greenhouse. Ectomycorrhizal development was monitored for up to 32 months. Seedling growth was assessed 2 years after inoculation. From 6 months after inoculation, Tuber melanosporum ectomycorrhizae were successfully produced on five Quercus species endemic to China, as shown by morphological, anatomical, and molecular analyses. Quercus mongolica and Q. longispica showed high receptivity to mycorrhization by T. melanosporum. The symbioses obtained with these two species and with Quercus senescens were stable for at least 32 months. Averaged over all three oak species, mycorrhization by T. melanosporum significantly enhanced canopy diameter, number of leaves, and mean leaf dimension. In spring 2019, mycorrhization by T. melanosporum accelerated budbreak in Q. mongolica. Quercus fabrei and Q. variabilis formed ectomycorrhizae up to 9 months after inoculation but seedlings died 3 months later, probably because of damage by grazing insects. Quercus pseudosemecarpifolia failed to form ectomycorrhizae. Results suggest that T. melanosporum–mycorrhized Q. mongolica and Q. longispica seedlings could be tested for ascocarp production and increased performance in the field.