The In Vitro Mass-Produced Model Mycorrhizal Fungus,Rhizophagus irregularis,Significantly Increases Yields of the Globally Important Food Security Crop Cassava

The arbuscular mycorrhizal symbiosis is formed between arbuscular mycorrhizal fungi (AMF) and plant roots. The fungi provide the plant with inorganic phosphate (P). The symbiosis can result in increased plant growth. Although most global food crops naturally form this symbiosis, very few studies have shown that their practical application can lead to large-scale increases in food production. Application of AMF to crops in the tropics is potentially effective for improving yields. However, a main problem of using AMF on a large-scale is producing cheap inoculum in a clean sterile carrier and sufficiently concentrated to cheaply transport. Recently, mass-produced in vitro inoculum of the model mycorrhizal fungus Rhizophagus irregularis became available, potentially making its use viable in tropical agriculture. One of the most globally important food plants in the tropics is cassava. We evaluated the effect of in vitro mass-produced R. irregularis inoculum on the yield of cassava crops at two locations in Colombia. A significant effect of R. irregularis inoculation on yield occurred at both sites. At one site, yield increases were observed irrespective of P fertilization. At the other site, inoculation with AMF and 50% of the normally applied P gave the highest yield. Despite that AMF inoculation resulted in greater food production, economic analyses revealed that AMF inoculation did not give greater return on investment than with conventional cultivation. However, the amount of AMF inoculum used was double the recommended dose and was calculated with European, not Colombian, inoculum prices. R. irregularis can also be manipulated genetically in vitro, leading to improved plant growth. We conclude that application of in vitro R. irregularis is currently a way of increasing cassava yields, that there is a strong potential for it to be economically profitable and that there is enormous potential to improve this efficiency further in the future.     

High effectiveness of exotic arbuscular mycorrhizal fungi is reflected in improved rhizobial symbiosis and trehalose turnover in Cajanus cajan genotypes grown under salinity stress

Arbuscular mycorrhizal fungi (AMF) improve functioning of legume-Rhizobium symbiosis under salinity. However, plant responses to mycorrhization vary depending on the plant and fungal species. The current study aimed to compare the effectiveness of a native inoculum from saline soil and two exotic isolates, Funneliformis mosseae and Rhizophagus irregularis on two Cajanus cajan (pigeonpea) genotypes (Paras, Pusa 2002) subjected to NaCl stress. Salinity depleted nodulation and nutrient status in both genotypes with higher negative effects in Paras. Although all AM fungi improved growth, R. irregularis performed better by promoting higher biomass accumulation, nodulation, N₂ fixation and N, P uptake which correlated with higher AM colonization. R. irregularis inoculated plants also accumulated higher trehalose in nodules due to decreased trehalase and increased trehalose-6-P synthase, trehalose-6-phosphatase activities. The results suggest that higher stability of R. irregularis-pigeonpea symbiosis under salt stress makes it an effective ameliorator for overcoming salt stress in pigeonpea.     

Glomeromycotan mycorrhizal fungi from tropical Australia III. Measuring diversity in natural and disturbed habitats

Aims and Background

The aim was to investigate the diversity and distribution of Glomeromycotan fungi forming arbuscular mycorrhizal associations (AMF) in undisturbed and disturbed habitats in the vicinity of Kakadu National Park in tropical Australia. This is a tropical region with a 7–9 month dry season and a monsoonal wet season. Complimentary methods of fungus detection were used to investigate the diversity and relative dominance of AMF at a regional scale.

Methods

Soils were sampled from 32 sites, representing eucalypt savanna woodlands, wetlands, sandstone escarpment, rainforest, and disturbed mine waste rock dumps (overburden or spoil). Populations of AMF were identified and quantified using spores from soil. Morphology patterns of fungi colonising bait plant roots were examined and isolates were obtained by four complimentary pot-culturing methods.

Results

Different methods of detecting fungi produced different answers about which AMF were most important in the tested soils. In particular, spore surveys apparently underestimated the importance of Glomus species and overestimated the activity of Acaulospora species with numerous small spores, while calculated spore biovolumes overestimated the importance of Scutellospora and Gigaspora species with large spores, relative to inoculum levels of these fungus categories measured in bioassays. Spore surveys revealed 15 species of fungi and 8 additional fungi were recovered from the same soil samples using pot-culture isolation methods. Pot-cultures were especially important for detecting Glomus species that had high inoculum levels, but rarely produced spores in soils. Spores of AMF increased in abundance as vegetation developed in mine habitats reaching a peak that was higher than in undisturbed plant communities. Spore numbers (but not biovolumes) were well correlated with bioassay measurements of inoculum levels.

Conclusions

Most AMF species were widespread, but several were restricted to disturbed habitats or wetland soils. Undisturbed sites had a substantially higher diversity of AMF than partially vegetated mine waste rock dumps. It is recommended that AMF population surveys should not be based entirely on spore occurrence data, to avoid overlooking important fungi that sporulate infrequently. These fungi could be detected by bioassays or pot culture isolation from soil. Major variations in the detectability of AMF correspond to different life history strategies and can mask variations in their abundance.     

<Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> MUCL 41833 can colonize and improve P uptake of <Emphasis Type="Italic">Plantago lanceolata</Emphasis> after exposure to ionizing gamma radiation in root organ culture

Long-lived radionuclides such as ⁹⁰Sr and ¹³⁷Cs can be naturally or accidentally deposited in the upper soil layers where they emit β/γ radiation. Previous studies have shown that arbuscular mycorrhizal fungi (AMF) can accumulate and transfer radionuclides from soil to plant, but there have been no studies on the direct impact of ionizing radiation on AMF. In this study, root organ cultures of the AMF Rhizophagus irregularis MUCL 41833 were exposed to 15.37, 30.35, and 113.03 Gy gamma radiation from a ¹³⁷Cs source. Exposed spores were subsequently inoculated to Plantago lanceolata seedlings in pots, and root colonization and P uptake evaluated. P. lanceolata seedlings inoculated with non-irradiated AMF spores or with spores irradiated with up to 30.35 Gy gamma radiation had similar levels of root colonization. Spores irradiated with 113.03 Gy gamma radiation failed to colonize P. lanceolata roots. P content of plants inoculated with non-irradiated spores or of plants inoculated with spores irradiated with up to 30.35 Gy gamma radiation was higher than in non-mycorrhizal plants or plants inoculated with spores irradiated with 113.03 Gy gamma radiation. These results demonstrate that spores of R. irregularis MUCL 41833 are tolerant to chronic ionizing radiation at high doses.     

Tomato belowground–aboveground interactions: <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> affects foraging behavior and life history traits of the predator <Emphasis Type="Italic">Macrolophus pygmaeus</Emphasis> (Hemiptera: Miridae)

In recent years, studies on arbuscular mycorrhizal fungi (AMF) have been revealing that the belowground symbiosis can influence the performance of aboveground herbivores and their natural enemies through its effects on the host plant. In this study, we tested whether the colonization of tomato plants by the arbuscular mycorrhizal fungus Rhizophagus irregularis (Syn. Glomus intraradices Schenk and Smith) (Glomeromycota: Glomeraceae) affects the performance of the zoophytophagous mirid bug Macrolophus pygmaeus Rambur (Hemiptera: Miridae). Mycorrhizal colonization in tomato plants positively influenced the predator host-plant acceptance for feeding and oviposition, as well as nymphal survival and female weight. We hypothesize that AMF can modify mirid bug foraging behavior and performance.     

Conserved and reproducible bacterial communities associate with extraradical hyphae of arbuscular mycorrhizal fungi

Extraradical hyphae (ERH) of arbuscular mycorrhizal fungi (AMF) extend from plant roots into the soil environment and interact with soil microbial communities. Evidence of positive and negative interactions between AMF and soil bacteria point to functionally important ERH-associated communities. To characterize communities associated with ERH and test controls on their establishment and composition, we utilized an in-growth core system containing a live soil–sand mixture that allowed manual extraction of ERH for 16S rRNA gene amplicon profiling. Across experiments and soils, consistent enrichment of members of the Betaproteobacteriales, Myxococcales, Fibrobacterales, Cytophagales, Chloroflexales, and Cellvibrionales was observed on ERH samples, while variation among samples from different soils was observed primarily at lower taxonomic ranks. The ERH-associated community was conserved between two fungal species assayed, Glomus versiforme and Rhizophagus irregularis, though R. irregularis exerted a stronger selection and showed greater enrichment for taxa in the Alphaproteobacteria and Gammaproteobacteria. A distinct community established within 14 days of hyphal access to the soil, while temporal patterns of establishment and turnover varied between taxonomic groups. Identification of a conserved ERH-associated community is consistent with the concept of an AMF microbiome and can aid the characterization of facilitative and antagonistic interactions influencing the plant-fungal symbiosis.Subject terms: Symbiosis, Microbiome     

Effects of Rhizophagus irregularis MUCL 41833 on the reproduction of Radopholus similis in banana plantlets grown under in vitro culture conditions

The role of arbuscular mycorrhizal fungi (AMF) in the control of migratory endoparasitic nematodes is nowadays largely admitted. Most studies were conducted under greenhouse conditions and a few used in vitro cultures with transgenic root organs. Here, we reported, for the first time, on the interaction between an AMF, Rhizophagus irregularis MUCL 41833 and Radopholus similis in roots of banana plantlets grown under in vitro culture conditions. The banana plantlets were pre-mycorrhized in an extraradical mycelium network arising from a Medicago truncatula donor seedling, before transfer to an autotrophic in vitro cultivation system and subsequent nematode inoculation. Both microorganisms were able to complete their life cycle in the absence as well as in presence of each other. The total R. similis population (i.e., summed over the roots and growth medium) as well as the surface of root necrosis was significantly reduced by 60 and 56 %, respectively, in the AMF-colonized banana plantlets. By contrast, nematodes had no visible impact on root colonization (i.e., percentage of arbuscules, intraradical spores/vesicles, and hyphae) by AMF and on the number of spores and hyphal length produced in the medium. These results clearly demonstrated that pre-mycorrhized banana plants could outcompete R. similis, while root colonization was not affected by the nematodes. They underline the interest of the novel in vitro cultivation system as a promising tool to investigate the biochemical factors and molecular mechanisms involved in the bio-protection conferred by AMF to a major root pathogen of banana.     

Identity and combinations of arbuscular mycorrhizal fungal isolates influence plant resistance and insect preference

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The impact of arbuscular mycorrhizal fungi on tomato plant resistance against Tuta absoluta (Meyrick) in greenhouse conditions

The symbiotic relationship between plants and arbuscular mycorrhizal fungi (AMF) improves plant growth and increases its resistance to pests and diseases. Mycorrhizal fungi are among the specialized fungi associated with the rhizosphere and are completely dependent on plant organic carbon. In this research tomato, Solanum lycopersicum L. was used as the host plant to evaluate the interaction effects between inoculation of tomato plant with AMF and feeding of tomato leaf miner, Tuta absoluta (Meyrick). In addition, plant growth parameters and growth rate of insect were assessed. The mycorrhizal treatment included a mixture of four fungal species (Funneliformis mosseae, Rhizophagus intraradices, R. irregularis and Glomus iranicus). The results of the experiment showed that tomato plant roots were well colonized (66.29%) by AMF and there was a significant mutual relationship between the insects feeding on the plants and the fungi. Feeding by the insects on plants inoculated with the fungus increased percentage of colonization by AMF in plants infested with the insect as compared to the control plants. The results also indicated that growth parameters and phosphorus content of the plants inoculated with fungi significantly increased compared to the control group. Moreover, significantly lower growth rate and consumption index observed in the T. absoluta larvae were fed on the leaves of plants treated with AMF compared to leaves of plants not inoculated with AMF.     

SeSaMe:Metagenome Sequence Classification of Arbuscular Mycorrhizal Fungi-associated Microorganisms

Arbuscular mycorrhizal fungi (AMF) are plant root symbionts that play key roles in plant growth and soil fertility. They are obligate biotrophic fungi that form coenocytic multinucleated hyphae and spores. Numerous studies have shown that diverse microorganisms live on the surface of and inside their mycelia, resulting in a metagenome when whole-genome sequencing (WGS) data are obtained from sequencing AMF cultivated in vivo. The metagenome contains not only the AMF sequences, but also those from associated microorganisms. In this study, we introduce a novel bioinformatics program, Spore-associated Symbiotic Microbes (SeSaMe), designed for taxonomic classification of short sequences obtained by next-generation DNA sequencing. A genus-specific usage bias database was created based on amino acid usage and codon usage of a three consecutive codon DNA 9-mer encoding an amino acid trimer in a protein secondary structure. The program distinguishes between coding sequence (CDS) and non-CDS, and classifies a query sequence into a genus group out of 54 genera used as reference. The mean percentages of correct predictions of the CDS and the non-CDS test sets at the genus level were 71% and 50% for bacteria, 68% and 73% for fungi (excluding AMF), and 49% and 72% for AMF (Rhizophagus irregularis), respectively. SeSaMe provides not only a means for estimating taxonomic diversity and abundance but also the gene reservoir of the reference taxonomic groups associated with AMF. Therefore, it enables users to study the symbiotic roles of associated microorganisms. It can also be applicable to other microorganisms as well as soil metagenomes. SeSaMe is freely available at www.fungalsesame.org.     

Isolate Identity Determines Plant Tolerance to Pathogen Attack in Assembled Mycorrhizal Communities

Arbuscular mycorrhizal fungi (AMF) are widespread soil microorganisms that associate mutualistically with plant hosts. AMF receive photosynthates from the host in return for various benefits. One of such benefits is in the form of enhanced pathogen tolerance. However, this aspect of the symbiosis has been understudied compared to effects on plant growth and its ability to acquire nutrients. While it is known that increased AMF species richness positively correlates with plant productivity, the relationship between AMF diversity and host responses to pathogen attack remains obscure. The objective of this study was to test whether AMF isolates can differentially attenuate the deleterious effects of a root pathogen on plant growth, whether the richest assemblage of AMF isolates provides the most tolerance against the pathogen, and whether AMF-induced changes to root architecture serve as a mechanism for improved plant disease tolerance. In a growth chamber study, we exposed the plant oxeye daisy (Leucanthemum vulgare) to all combinations of three AMF isolates and to the plant root pathogen Rhizoctonia solani. We found that the pathogen caused an 81% reduction in shoot and a 70% reduction in root biomass. AMF significantly reduced the highly deleterious effect of the pathogen. Mycorrhizal plants infected with the pathogen produced 91% more dry shoot biomass and 72% more dry root biomass relative to plants solely infected with R. solani. AMF isolate identity was a better predictor of AMF-mediated host tolerance to the pathogen than AMF richness. However, the enhanced tolerance response did not result from AMF-mediated changes to root architecture. Our data indicate that AMF communities can play a major role in alleviating host pathogen attack but this depends primarily on the capacity of individual AMF isolates to provide this benefit.     

The extraradical proteins of Rhizophagus irregularis: A shotgun proteomics approach

Arbuscular Mycorrhizal fungi (AMF, Glomeromycota) form obligate symbiotic associations with the roots of most terrestrial plants. Our understanding of the molecular mechanisms enabling AMF propagation and AMF-host interaction is currently incomplete. Analysis of AMF proteomes could yield important insights and generate hypotheses on the nature and mechanism of AMF-plant symbiosis. Here, we examined the extraradical mycelium proteomic profile of the arbuscular mycorrhizal fungus Rhizophagus irregularis grown on Ri T-DNA transformed Chicory roots in a root organ culture setting. Our analysis detected 529 different peptides that mapped to 474 translated proteins in the R. irregularis genome. R. irregularis proteome was characterized by a high proportion of proteins (9.9 % of total, 21.4 % of proteins with functional prediction) mediating a wide range of signal transduction processes, e.g. Rho1 and Bmh2, Ca-signaling (calmodulin, and Ca channel protein), mTOR signaling (MAP3K7, and MAPKAP1), and phosphatidate signaling (phospholipase D1/2) proteins, as well as members of the Ras signaling pathway. In addition, the proteome contained an unusually large proportion (53.6 %) of hypothetical proteins, the majority of which (85.8 %) were Glomeromycota-specific. Forty-eight proteins were predicted to be surface/membrane associated, including multiple hypothetical proteins of yet-unrecognized functions. However, no evidence for the overproduction of specific proteins, previously implicated in promoting soil health and aggregation was obtained. Finally, the comparison of R. irregularis proteome to previously published AMF proteomes identified a core set of pathways and processes involved in AMF growth. We conclude that R. irregularis growth on chicory roots requires the activation of a wide range of signal transduction pathways, the secretion of multiple novel hitherto unrecognized Glomeromycota-specific proteins, and the expression of a wide array of surface-membrane associated proteins for cross kingdom cell-to-cell communications.     

Arbuscular mycorrhizal fungi reduce growth and infect roots of the non‐host plant Arabidopsis thaliana

The arbuscular mycorrhizal (AM) symbiosis is widespread throughout the plant kingdom and important for plant nutrition and ecosystem functioning. Nonetheless, most terrestrial ecosystems also contain a considerable number of non‐mycorrhizal plants. The interaction of such non‐host plants with AM fungi (AMF) is still poorly understood. Here, in three complementary experiments, we investigated whether the non‐mycorrhizal plant Arabidopsis thaliana, the model organism for plant molecular biology and genetics, interacts with AMF. We grew A. thaliana alone or together with a mycorrhizal host species (either Trifolium pratense or Lolium multiflorum) in the presence or absence of the AMF Rhizophagus irregularis. Plants were grown in a dual‐compartment system with a hyphal mesh separating roots of A. thaliana from roots of the host species, avoiding direct root competition. The host plants in the system ensured the presence of an active AM fungal network. AM fungal networks caused growth depressions in A. thaliana of more than 50% which were not observed in the absence of host plants. Microscopy analyses revealed that R. irregularis supported by a host plant was capable of infecting A. thaliana root tissues (up to 43% of root length colonized), but no arbuscules were observed. The results reveal high susceptibility of A. thaliana to R. irregularis, suggesting that A. thaliana is a suitable model plant to study non‐host/AMF interactions and the biological basis of AM incompatibility.     

Arbuscular mycorrhizal fungal communities along a pedo-hydrological gradient in a Central Amazonian terra firme forest

Little attention has been paid to plant mutualistic interactions in the Amazon rainforest, and the general pattern of occurrence and diversity of arbuscular mycorrhizal fungi (AMF) in these ecosystems is largely unknown. This study investigated AMF communities through their spores in soil in a ‘terra firme forest’ in Central Amazonia. The contribution played by abiotic factors and plant host species identity in regulating the composition, abundance and diversity of such communities along a topographic gradient with different soils and hydrology was also evaluated. Forty-one spore morphotypes were observed with species belonging to the genera Glomus and Acaulospora, representing 44 % of the total taxa. Soil texture and moisture, together with host identity, were predominant factors responsible for shaping AMF communities along the pedo-hydrological gradient. However, the variability within AMF communities was largely associated with shifts in the relative abundance of spores rather than changes in species composition, confirming that common AMF species are widely distributed in plant communities and all plants recruited into the forest are likely to be exposed to the dominant sporulating AMF species.     

Symbiosis of Arbuscular Mycorrhizal Fungi and Robinia pseudoacacia L. Improves Root Tensile Strength and Soil Aggregate Stability

Robinia pseudoacacia L. (black locust) is a widely planted tree species on Loess Plateau for revegetation. Due to its symbiosis forming capability with arbuscular mycorrhizal (AM) fungi, we explored the influence of arbuscular mycorrhizal fungi on plant biomass, root morphology, root tensile strength and soil aggregate stability in a pot experiment. We inoculated R. pseudoacacia with/without AM fungus (Rhizophagus irregularis or Glomus versiforme), and measured root colonization, plant growth, root morphological characters, root tensile force and tensile strength, and parameters for soil aggregate stability at twelve weeks after inoculation. AM fungi colonized more than 70% plant root, significantly improved plant growth. Meanwhile, AM fungi elevated root morphological parameters, root tensile force, root tensile strength, Glomalin-related soil protein (GRSP) content in soil, and parameters for soil aggregate stability such as water stable aggregate (WSA), mean weight diameter (MWD) and geometric mean diameter (GMD). Root length was highly correlated with WSA, MWD and GMD, while hyphae length was highly correlated with GRSP content. The improved R. pseudoacacia growth, root tensile strength and soil aggregate stability indicated that AM fungi could accelerate soil fixation and stabilization with R. pseudoacacia, and its function in revegetation on Loess Plateau deserves more attention.     

Arbuscular mycorrhizal fungi enhance spotted knapweed growth across a riparian chronosequence

Arbuscular mycorrhizal fungi (AMF) mediate nutrient uptake that accelerates plant growth and reproduction. Thus, AMF may promote plant invasions often observed along rivers. We assessed the importance of AMF in improving growth of the invasive species, spotted knapweed (Centaurea stoebe), during succession of riparian vegetation along a flood plain in Montana, USA. We grew spotted knapweed with and without AMF in soils collected from riparian sites ranging from 1 to 72 years old and measured the plant’s growth response to AMF. We observed variability in relative effects of AMF, with greatest growth benefits in recently deposited alluvial sediments. We then separated effects of soil and inoculum source by growing spotted knapweed with soils and inocula collected from young or old sites and found that growth responses were greatest in young soils regardless of inoculum source. Our results demonstrate that AMF directly benefit growth of spotted knapweed, especially in soils that typify early successional sites on this alluvial flood plain.     

Arbuscular mycorrhizal fungi improve the growth and drought tolerance of Cinnamomum migao by enhancing physio‐biochemical responses

Drought is the main limiting factor for plant growth in karst areas with a fragile ecological environment. Cinnamomum migao H.W. Li is an endemic medicinal woody plant present in the karst areas of southwestern China, and it is endangered due to poor drought tolerance. Arbuscular mycorrhizal fungi (AMF) are known to enhance the drought tolerance of plants. However, few studies have examined the contribution of AMF in improving the drought tolerance of C. migao seedlings. Therefore, we conducted a series of experiments to determine whether a single inoculation and coinoculation of AMF (Claroideoglomus lamellosum and Claroideoglomus etunicatum) enhanced the drought tolerance of C. migao. Furthermore, we compared the effects of single inoculation and coinoculation with different inoculum sizes (20, 40, 60, and 100 g; four replicates per treatment) on mycorrhizal colonization rate, plant growth, photosynthetic parameters, antioxidant enzyme activity, and malondialdehyde (MDA) and osmoregulatory substance contents. The results showed that compared with nonmycorrhizal plants, AMF colonization significantly improved plant growing status; net photosynthetic rate; superoxide dismutase, catalase, and peroxidase activities; and soluble sugar, soluble protein, and proline contents. Furthermore, AMF colonization increased relative water content and reduced MDA content in cells. These combined cumulative effects of AMF symbiosis ultimately enhanced the drought tolerance of seedlings and were closely related to the inoculum size. With an increase in inoculum size, the growth rate and drought tolerance of plants first increased and then decreased. The damage caused by drought stress could be reduced by inoculating 40–60 g of AMF, and the effect of coinoculation was significantly better than that of single inoculation at 60 g of AMF, while the effect was opposite at 40 g of AMF. Additionally, the interaction between AMF and inoculum sizes had a significant effect on drought tolerance. In conclusion, the inoculation of the AMF (Cl. lamellosum and Cl. etunicatum) improved photosynthesis, activated antioxidant enzymes, regulated cell osmotic state, and enhanced the drought tolerance of C. migao, enabling its growth in fragile ecological environments.     

Identification and Isolation of Two Ascomycete Fungi from Spores of the Arbuscular Mycorrhizal Fungus Scutellospora castanea

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.