A tripartite bacterial-fungal-plant symbiosis in the mycorrhiza-shaped microbiome drives plant growth and mycorrhization.

BACKGROUND: Plant microbiomes play crucial roles in nutrient cycling and plant growth, and are shaped by a complex interplay between plants, microbes, and the environment. The role of bacteria as mediators of the 400-million-year-old partnership between the majority of land plants and, arbuscular mycorrhizal (AM) fungi is still poorly understood. Here, we test whether AM hyphae-associated bacteria influence the success of the AM symbiosis. RESULTS: Using partitioned microcosms containing field soil, we discovered that AM hyphae and roots selectively assemble their own microbiome from the surrounding soil. In two independent experiments, we identified several bacterial genera, including Devosia, that are consistently enriched on AM hyphae. Subsequently, we isolated 144 pure bacterial isolates from a mycorrhiza-rich sample of extraradical hyphae and isolated Devosia sp. ZB163 as root and hyphal colonizer. We show that this AM-associated bacterium synergistically acts with mycorrhiza on the plant root to strongly promote plant growth, nitrogen uptake, and mycorrhization. CONCLUSIONS: Our results highlight that AM fungi do not function in  isolation and that the plant-mycorrhiza symbiont can recruit beneficial bacteria that support the symbiosis. Video Abstract.

Mycorrhizas drive the evolution of plant adaptation to drought.

Plant adaptation to drought facilitates major ecological transitions, and will likely play a vital role under looming climate change. Mycorrhizas, i.e. strategic associations between plant roots and soil-borne symbiotic fungi, can exert strong influence on the tolerance to drought of extant plants. Here, I show how mycorrhizal strategy and drought adaptation have been shaping one another throughout the course of plant evolution. To characterize the evolutions of both plant characters, I applied a phylogenetic comparative method using data of 1,638 extant species globally distributed. The detected correlated evolution unveiled gains and losses of drought tolerance occurring at faster rates in lineages with ecto- or ericoid mycorrhizas, which were on average about 15 and 300 times faster than in lineages with the arbuscular mycorrhizal and naked root (non-mycorrhizal alone or with facultatively arbuscular mycorrhizal) strategy, respectively. My study suggests that mycorrhizas can play a key facilitator role in the evolutionary processes of plant adaptation to critical changes in water availability across global climates.

Mycelium chemistry differs markedly between ectomycorrhizal and arbuscular mycorrhizal fungi.

The chemical quality of soil carbon (C) inputs is a major factor controlling litter decomposition and soil C dynamics. Mycorrhizal fungi constitute one of the dominant pools of soil microbial C, while their litter quality (chemical proxies of litter decomposability) is understood poorly, leading to major uncertainties in estimating soil C dynamics. We examined litter decomposability of arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungal species using samples obtained from in vitro cultivation. We showed that the chemical composition of AM and EM fungal mycelium differs significantly: EM fungi have higher concentrations of labile (water-soluble, ethanol-soluble) and recalcitrant (non-extractable) chemical components, while AM fungi have higher concentrations of acid-hydrolysable components. Our results imply that differences in decomposability traits among mycorrhizal fungal guilds represent a critically important driver of the soil C cycle, which could be as vital as is recognized for differences among aboveground plant litter.

A coumarin exudation pathway mitigates arbuscular mycorrhizal incompatibility in Arabidopsis thaliana.

KEY MESSAGE: Overexpression of genes involved in coumarin production and secretion can mitigate mycorrhizal incompatibility in nonhost Arabidopsis plants. The coumarin scopoletin, in particular, stimulates pre-penetration development and metabolism in mycorrhizal fungi. Although most plants can benefit from mutualistic associations with arbuscular mycorrhizal (AM) fungi, nonhost plant species such as the model Arabidopsis thaliana have acquired incompatibility. The transcriptional response of Arabidopsis to colonization by host-supported AM fungi switches from initial AM recognition to defense activation and plant growth antagonism. However, detailed functional information on incompatibility in nonhost-AM fungus interactions is largely missing. We studied interactions between host-sustained AM fungal networks of Rhizophagus irregularis and 18 Arabidopsis genotypes affected in nonhost penetration resistance, coumarin production and secretion, and defense (salicylic acid, jasmonic acid, and ethylene) and growth hormones (auxin, brassinosteroid, cytokinin, and gibberellin). We demonstrated that root-secreted coumarins can mitigate incompatibility by stimulating fungal metabolism and promoting initial steps of AM colonization. Moreover, we provide evidence that major molecular defenses in Arabidopsis do not operate as primary mechanisms of AM incompatibility nor of growth antagonism. Our study reveals that, although incompatible, nonhost plants can harbor hidden tools that promote initial steps of AM colonization. Moreover, it uncovered the coumarin scopoletin as a novel signal in the pre-penetration dialogue, with possible implications for the chemical communication in plant-mycorrhizal fungi associations.

A Stimulatory Role for Cytokinin in the Arbuscular Mycorrhizal Symbiosis of Pea.

The arbuscular mycorrhizal (AM) symbiosis between terrestrial plants and AM fungi is regulated by plant hormones. For most of these, a role has been clearly assigned in this mutualistic interaction; however, there are still contradictory reports for cytokinin (CK). Here, pea plants, the wild type (WT) cv. Sparkle and its mutant E151 (Pssym15), were inoculated with the AM fungus Rhizophagus irregularis. E151 has previously been characterized as possessing high CK levels in non-mycorrhizal (myc-) roots and exhibiting high number of fungal structures in mycorrhizal (myc+) roots. Myc- and myc+ plants were treated 7, 9, and 11 days after inoculation (DAI) with synthetic compounds known to alter CK status. WT plants were treated with a synthetic CK [6-benzylaminopurine (BAP)] or the CK degradation inhibitor INCYDE, whereas E151 plants were treated with the CK receptor antagonist PI-55. At 13 DAI, plant CK content was analyzed by mass spectrometry. The effects of the synthetic compounds on AM colonization were assessed at 28 (WT) or 35 (E151) DAI via a modified magnified intersections method. The only noticeable difference seen between myc- and myc+ plants in terms of CK content was in the levels of nucleotides (NTs). Whereas WT plants responded to fungi by lowering their NT levels, E151 plants did not. Since NTs are thought to be converted into active CK forms, this result suggests that active CKs were synthesized more effectively in WT than in E151. In general, myc+ and myc- WT plants responded similarly to INCYDE by lowering significantly their NT levels and increasing slightly their active CK levels; these responses were less obvious in BAP-treated WT plants. In contrast, the response of E151 plants to PI-55 depended on the plant mycorrhizal status. Whereas treated myc- plants exhibited high NT and low active CK levels, treated myc+ plants displayed low levels of both NTs and active CKs. Moreover, treated WT plants were more colonized than treated E151 plants. We concluded that CKs have a stimulatory role in AM colonization because increased active CK levels were paralleled with increased AM colonization while decreased CK levels corresponded to reduced AM colonization.

Molecular dialogue between arbuscular mycorrhizal fungi and the nonhost plant Arabidopsis thaliana switches from initial detection to antagonism.

Approximately 29% of all vascular plant species are unable to establish an arbuscular mycorrhizal (AM) symbiosis. Despite this, AM fungi (Rhizophagus spp.) are enriched in the root microbiome of the nonhost Arabidopsis thaliana, and Arabidopsis roots become colonized when AM networks nurtured by host plants are available. Here, we investigated the nonhost-AM fungus interaction by analyzing transcriptional changes in Rhizophagus, Arabidopsis and the host plant Medicago truncatula while growing in the same mycorrhizal network. In early interaction stages, Rhizophagus activated the Arabidopsis strigolactone biosynthesis genes CCD7 and CCD8, suggesting that detection of AM fungi is not completely impaired. However, in colonized Arabidopsis roots, fungal nutrient transporter genes GintPT, GintAMT2, GintMST2 and GintMST4, essential for AM symbiosis, were not activated. RNA-seq transcriptome analysis pointed to activation of costly defenses in colonized Arabidopsis roots. Moreover, Rhizophagus colonization caused a 50% reduction in shoot biomass, but also led to enhanced systemic immunity against Botrytis cinerea. This suggests that early signaling between AM fungi and Arabidopsis is not completely impaired and that incompatibility appears at later interaction stages. Moreover, Rhizophagus-mediated defenses coincide with reduced Arabidopsis growth, but also with systemic disease resistance, highlighting the multifunctional role of AM fungi in host and nonhost interactions.

Belowground Inoculation With Arbuscular Mycorrhizal Fungi Increases Local and Systemic Susceptibility of Rice Plants to Different Pest Organisms.

Plants face numerous challenges from both aboveground and belowground stressors, and defend themselves against harmful insects and microorganisms in many ways. Because plant responses to biotic stresses are not only local but also systemic, belowground interactions can influence aboveground interactions in both natural and agricultural ecosystems. Arbuscular mycorrhizal fungi (AMF) are soilborne organisms that form symbiotic associations with many plant roots and are thought to play a central role in plant nutrition, growth, and fitness. In the present study, we focused on the influence of AMF on rice defense against pests. We inoculated rice plants with AMF in several field and greenhouse experiments to test whether the interaction of AMF with rice roots changes the resistance of rice against two chewing insects, the rice water weevil (Lissorhoptrus oryzophilus Kuschel, RWW) and the fall armyworm (Spodoptera frugiperda, FAW), and against infection by sheath blight (Rhizoctonia solani, ShB). Both in field and greenhouse experiments, the performance of insects and the pathogen on rice was enhanced when plants were inoculated with AMF. In the field, inoculating rice plants with AMF resulted in higher numbers of RWW larvae on rice roots. In the greenhouse, more RWW first instars emerged from AMF-colonized rice plants than from non-colonized control plants. Weight gains of FAW larvae were higher on rice plants treated with AMF inoculum. Lesion lengths and susceptibility to ShB infection were higher in rice plants colonized by AMF. Although AMF inoculation enhanced the growth of rice plants, the nutritional analyses of root and shoot tissues indicated no major increases in the concentrations of nutrients in rice plants colonized by AMF. The large effects on rice susceptibility to pests in the absence of large effects on plant nutrition suggest that AMF colonization influences other mechanisms of susceptibility (e.g., defense signaling processes). This study represents the first study conducted in the U.S. in rice showing AMF-induced plant susceptibility to several antagonists that specialize on different plant tissues. Given the widespread occurrence of AMF, our findings will help to provide a different perspective into the causal basis of rice systemic resistance/susceptibility to insects and pathogens.

Non-Mycorrhizal Plants: The Exceptions that Prove the Rule.

The widespread symbiotic interaction between plants and arbuscular mycorrhizal (AM) fungi relies on a complex molecular dialog with reciprocal benefits in terms of nutrition, growth, and protection. Approximately 29% of all vascular plant species do not host AM symbiosis, including major crops. Under certain conditions, however, presumed non-host plants can become colonized by AM fungi and develop rudimentary AM (RAM) phenotypes. Here we zoom in on the mustard family (Brassicaceae), which harbors AM hosts, non-hosts, and presumed non-host species such as Arabidopsis thaliana, for which conditional RAM colonization has been described. We advocate that RAM phenotypes and redundant genomic elements of the symbiotic 'toolkit' are missing links that can help to unravel genetic constraints that drive the evolution of symbiotic incompatibility.

Shoot- and root-borne cytokinin influences arbuscular mycorrhizal symbiosis.

The arbuscular mycorrhizal (AM) symbiosis is functionally important for the nutrition and growth of most terrestrial plants. Nearly all phytohormones are employed by plants to regulate the symbiosis with AM fungi, but the regulatory role of cytokinin (CK) is not well understood. Here, we used transgenic tobacco (Nicotiana tabacum) with a root-specific or constitutive expression of CK-degrading CKX genes and the corresponding wild-type to investigate whether a lowered content of CK in roots or in both roots and shoots influences the interaction with the AM fungus Rhizophagus irregularis. Our data indicates that shoot CK has a positive impact on AM fungal development in roots and on the root transcript level of an AM-responsive phosphate transporter gene (NtPT4). A reduced CK content in roots caused shoot and root growth depression following AM colonization, while neither the uptake of phosphorus or nitrogen nor the root transcript levels of NtPT4 were significantly affected. This suggests that root CK may restrict the C availability from the roots to the fungus thus averting parasitism by AM fungi. Taken together, our study indicates that shoot- and root-borne CK have distinct roles in AM symbiosis. We propose a model illustrating how plants may employ CK to regulate nutrient exchange with the ubiquitous AM fungi.

A fungal endophyte helps plants to tolerate root herbivory through changes in gibberellin and jasmonate signaling.

Plant-microbe mutualisms can improve plant defense, but the impact of root endophytes on below-ground herbivore interactions remains unknown. We investigated the effects of the root endophyte Piriformospora indica on interactions between rice (Oryza sativa) plants and its root herbivore rice water weevil (RWW; Lissorhoptrus oryzophilus), and how plant jasmonic acid (JA) and GA regulate this tripartite interaction. Glasshouse experiments with wild-type rice and coi1-18 and Eui1-OX mutants combined with nutrient, jasmonate and gene expression analyses were used to test: whether RWW adult herbivory above ground influences subsequent damage caused by larval herbivory below ground; whether P. indica protects plants against RWW; and whether GA and JA signaling mediate these interactions. The endophyte induced plant tolerance to root herbivory. RWW adults and larvae acted synergistically via JA signaling to reduce root growth, while endophyte-elicited GA biosynthesis suppressed the herbivore-induced JA in roots and recovered plant growth. Our study shows for the first time the impact of a root endophyte on plant defense against below-ground herbivores, adds to growing evidence that induced tolerance may be an important root defense, and implicates GA as a signal component of inducible plant tolerance against biotic stress.

Induced jasmonate signaling leads to contrasting effects on root damage and herbivore performance.

Induced defenses play a key role in plant resistance against leaf feeders. However, very little is known about the signals that are involved in defending plants against root feeders and how they are influenced by abiotic factors. We investigated these aspects for the interaction between rice (Oryza sativa) and two root-feeding insects: the generalist cucumber beetle (Diabrotica balteata) and the more specialized rice water weevil (Lissorhoptrus oryzophilus). Rice plants responded to root attack by increasing the production of jasmonic acid (JA) and abscisic acid, whereas in contrast to in herbivore-attacked leaves, salicylic acid and ethylene levels remained unchanged. The JA response was decoupled from flooding and remained constant over different soil moisture levels. Exogenous application of methyl JA to the roots markedly decreased the performance of both root herbivores, whereas abscisic acid and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid did not have any effect. JA-deficient antisense 13-lipoxygenase (asLOX) and mutant allene oxide cyclase hebiba plants lost more root biomass under attack from both root herbivores. Surprisingly, herbivore weight gain was decreased markedly in asLOX but not hebiba mutant plants, despite the higher root biomass removal. This effect was correlated with a herbivore-induced reduction of sucrose pools in asLOX roots. Taken together, our experiments show that jasmonates are induced signals that protect rice roots from herbivores under varying abiotic conditions and that boosting jasmonate responses can strongly enhance rice resistance against root pests. Furthermore, we show that a rice 13-lipoxygenase regulates root primary metabolites and specifically improves root herbivore growth.

Arbuscular mycorrhizal fungi affect glucosinolate and mineral element composition in leaves of Moringa oleifera.

Moringa is a mycorrhizal crop cultivated in the tropics and subtropics and appreciated for its nutritive and health-promoting value. As well as improving plant mineral nutrition, arbuscular mycorrhizal fungi (AMF) can affect plant synthesis of compounds bioactive against chronic diseases in humans. Rhizophagus intraradices and Funneliformis mosseae were used in a full factorial experiment to investigate the impact of AMF on the accumulation of glucosinolates, flavonoids, phenolic acids, carotenoids, and mineral elements in moringa leaves. Levels of glucosinolates were enhanced, flavonoids and phenolic acids were not affected, levels of carotenoids (including provitamin A) were species-specifically reduced, and mineral elements were affected differently, with only Cu and Zn being increased by the AMF. This study presents novel results on AMF effects on glucosinolates in leaves and supports conclusions that the impacts of these fungi on microelement concentrations in edible plants are species dependent. The nonspecific positive effects on glucosinolates and the species-specific negative effects on carotenoids encourage research on other AMF species to achieve general benefits on bioactive compounds in moringa.

Effect of arbuscular mycorrhizal fungi (Glomus intraradices) on the oviposition of rice water weevil (Lissorhoptrus oryzophilus).

Root-feeding insects are important drivers in ecosystems, and links between aboveground oviposition preference and belowground larval performance have been suggested. The root-colonizing arbuscular mycorrhizal fungi (AMF) play a central role in plant nutrition and are known to change host quality for root-feeding insects. However, it is not known if and how AMF affect the aboveground oviposition of insects whose offspring feed on roots. According to the preference-performance hypothesis, insect herbivores oviposit on plants that will maximize offspring performance. In a greenhouse experiment with rice (Oryza sativa), we investigated the effects of AMF (Glomus intraradices) on aboveground oviposition of rice water weevil (Lissorhoptrus oryzophilus), the larvae of which feed belowground on the roots. Oviposition (i.e., the numbers of eggs laid by weevil females in leaf sheaths) was enhanced when the plants were colonized by AMF. However, the leaf area consumed by adult weevils was not affected. Although AMF reduced plant biomass, it increased nitrogen (N) and phosphorus concentrations in leaves and N in roots. The results suggest that rice water weevil females are able to discriminate plants for oviposition depending on their mycorrhizal status. The discrimination is probably related to AMF-mediated changes in plant quality, i.e., the females choose to oviposit more on plants with higher nutrient concentrations to potentially optimize offspring performance. AMF-mediated change in plant host choice for chewing insect oviposition is a novel aspect of below- and aboveground interactions.