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.

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.

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.

Mycorrhizae for a sustainable world—the hot topic for a tropical ICOM

Are fungi from adult orchid roots the best symbionts at germination? A case study Abstract We studied mycobionts from advanced seedlings and adult mycorrhizal roots of the terrestrial orchid Arundina graminifolia. Fungi were isolated, identified by ITS sequencing, and tested for their impact on seed germination, protocorm formation, and development of advanced seedlings (emergence of first leaf) in vitro. Among the six fungal species isolated, four were not standard orchid mycorrhizal fungi (Fusarium solani, Cylindrocarpon sp., Acremonium sp., and Phlebiopsis flavidoalba) and did not support germination beyond imbibition and greening of the seeds during a span of 35 days. Over the same time, one Tulasnella species isolated from adult mycorrhiza allowed protocorm formation but not further development. However, another Tulasnella species isolated from advanced seedlings facilitated development to the advanced seedling stage. Our results support (i) the inability of occasional orchid root colonizers to support late seed germination, and (ii) the growing literature showing that fungal associates can change over orchid development. Functionally, we show that mycorrhizal taxa isolated from advanced seedlings can be more efficient than those from adults in supporting germination in some species, leading to recommendations for ex situ orchid conservation.

Initial microbial status modulates mycorrhizal inoculation effect on rhizosphere microbial communities Abstract Arbuscular mycorrhizal fungi (AMF) play a central role in rhizosphere functioning as they interact with both plants and soil microbial communities. The conditions in which AMF modify plant physiology and microbial communities in the rhizosphere are still poorly understood. In the present study, four different plant species, (clover, alfalfa, ryegrass, tall fescue) were cultivated in either sterilized (γ ray) or non-sterilized soil and either inoculated with a commercial AMF (Glomus LPA Val 1.) or not. After 20 weeks of cultivation, the mycorrhizal rate and shoot and root biomasses were measured. The abundance and composition of bacteria, archaea, and fungi were analyzed, respectively, by quantitative PCR (qPCR) and fingerprinting techniques. Whilst sterilization did not change the AMF capacity to modify plant biomass, significant changes in microbial communities were observed, depending on the taxon and the associated plant. AMF inoculation decreases both bacterial and archaeal abundance and diversity, with a greatest extent in sterilized samples. These results also show that AMF exert different selections on soil microbial communities according to the plant species they are associated with. This study suggests that the initial abundance and diversity of rhizosphere microbial communities should be considered when introducing AMF to cultures.

Mycorrhizal frequency, physiological parameters, and yield of strawberry plants inoculated with endomycorrhizal fungi and rhizosphere bacteria Abstract Due to the impoverishment of agricultural and horticultural soils and replant diseases, there is a need to use bioproducts and beneficial microorganisms in order to improve the quality of soils and growth substrates. For this reason, research was undertaken to assess the impact of arbuscular mycorrhizal fungi and rhizosphere bacteria on changes in soil microbiology, the degree of colonization of plant roots by mycorrhizal fungi, selected physiological parameters, and fruit quality and yield of the strawberry cultivar “Rumba.” The plants were inoculated with the mycorrhizal preparation Mykoflor (Rhizophagus irregularis, Funneliformis mosseae, Claroideoglomus etunicatum), MYC 800 (Rhizophagus intraradices), and the bacterial preparation Rhizocell C (Bacillus amyloliquefaciens IT45). The applied preparations increased the total number of bacteria and fungi in the soil and mycorrhizal frequency in the roots of the strawberry plants. They increased the chlorophyll “a” and total chlorophyll concentrations in the leaves as well as the rate of transpiration and CO2 concentration in the intercellular spaces in the leaves. The plants treated with Rhizocell C and MYC 800 exhibited a higher CO2 assimilation rate than control plants. The biopreparations increased chlorophyll fluorescence parameters such as maximum fluorescence (FM) and the maximum potential photochemical reaction efficiency in PS II (FV/FM). The influence of the species of rhizosphere bacteria and mycorrhizal fungi used in the experiment on the physiological traits of strawberry plants contributed, especially in the second year of the study, to increase the yield and mean weight of strawberry fruit.

Holobiont chronobiology: mycorrhiza may be a key to linking aboveground and underground rhythms Abstract Circadian clocks are nearly ubiquitous timing mechanisms that can orchestrate rhythmic behavior and gene expression in a wide range of organisms. Clock mechanisms are becoming well understood in fungal, animal, and plant model systems, yet many of these organisms are surrounded by a complex and diverse microbiota which should be taken into account when examining their biology. Of particular interest are the symbiotic relationships between organisms that have coevolved over time, forming a unit called a holobiont. Several studies have now shown linkages between the circadian rhythms of symbiotic partners. Interrelated regulation of holobiont circadian rhythms seems thus important to coordinate shifts in activity over the day for all the partners. Therefore, we suggest that the classical view of “chronobiological individuals” should include “a holobiont” rather than an organism. Unfortunately, mechanisms that may regulate interspecies temporal acclimation and the evolution of the circadian clock in holobionts are far from being understood. For the plant holobiont, our understanding is particularly limited. In this case, the holobiont encompasses two different ecosystems, one above and the other below the ground, with the two potentially receiving timing information from different synchronizing signals (Zeitgebers). The arbuscular mycorrhizal (AM) symbiosis, formed by plant roots and fungi, is one of the oldest and most widespread associations between organisms. By mediating the nutritional flux between the plant and the many microbes in the soil, AM symbiosis constitutes the backbone of the plant holobiont. Even though the importance of the AM symbiosis has been well recognized in agricultural and environmental sciences, its circadian chronobiology remains almost completely unknown. We have begun to study the circadian clock of arbuscular mycorrhizal fungi, and we compile and here discuss the available information on the subject. We propose that analyzing the interrelated temporal organization of the AM symbiosis and determining its underlying mechanisms will advance our understanding of the role and coordination of circadian clocks in holobionts in general.

Repeated fruiting of Japanese golden chanterelle in pot culture with host seedlings Abstract Yellow chanterelles are among the most popular wild edible ectomycorrhizal mushrooms worldwide. The representative European golden chanterelle, Cantharellus cibarius, has only once been reported to fruit under greenhouse conditions, due to the difficulty of establishing pure culture. Recently, we developed a new technique for establishing a pure culture of a Japanese golden chanterelle (Cantharellus anzutake), and conducted in vitro ectomycorrhizal synthesis using established strains and Pinus densiflora. Acclimated pine mycorrhizal seedlings colonized with C. anzutake in a pot system under laboratory conditions produced small but distinct basidiomata with developed basidiospores. C. anzutake mycorrhizae were established on Quercus serrata seedlings by inoculation of mycorrhizal root tips of the fungus synthesized on P. densiflora. A scaled-up C. anzutake–host system in larger pots (4 L soil volume) exhibited repeated fruiting at 20–24 °C under continuous light illumination at 150 μmol m−2 s−1 during a 2-year incubation period. Therefore, a C. anzutake cultivation trial is practical under controlled environmental conditions.

Regulation of the leaf proteome by inoculation of Populus × canescens with two Paxillus involutus isolates differing in root colonization rates Abstract During ectomycorrhizal symbioses, up to 30% of the carbon produced in leaves may be translocated to the fungal partner. Given that the leaf response to root colonization is largely unknown, we performed a leaf proteome analysis of Populus × canescens inoculated in vitro with two isolates of Paxillus involutus significantly differing in root colonization rates (65 ± 7% vs 14 ± 7%), together with plant growth and leaf biochemistry analyses to determine the response of plant leaves to ectomycorrhizal root colonization. The isolate that more efficiently colonized roots (isolate H) affected 9.1% of the leaf proteome compared with control plants. Simultaneously, ectomycorrhiza in isolate H-inoculated plants led to improved plant growth and an increased abundance of leaf proteins involved in protein turnover, stress response, carbohydrate metabolism, and photosynthesis. The protein increment was also correlated with increases in chlorophyll, foliar carbon, and carbohydrate contents. Although inoculation of P. × canescens roots with the other P. involutus isolate (isolate L, characterized by a low root colonization ratio) affected 6.8% of the leaf proteome compared with control plants, most proteins were downregulated. The proteomic signals of increased carbohydrate biosynthesis were not detected, and carbohydrate, carbon, and leaf pigment levels and plant biomass did not differ from the noninoculated plants. Our results revealed that the upregulation of the photosynthetic protein abundance and levels of leaf carbohydrate are positively related to rates of root colonization. Upregulation of photosynthetic proteins, chlorophyll, and leaf carbohydrate levels in ectomycorrhizal plants was positively related to root colonization rates and resulted in increased carbon translocation and sequestration underground.