Arbuscular Mycorrhizal Fungus Gigaspora Margarita Research Articles

Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita.

More than 70% of vascular plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. In addition to other effects this symbiosis is known for its significance for plant nutrition including iron. Fungal iron mobilization from soil is commonly dependent on siderophores. This study reports on a search for such iron-chelators in root tissue of Tagetes patula nana var. plena colonized by Gigaspora margarita. The AM colonized plants and uninoculated controls were grown under strictly axenic conditions. HPLC analyses of aqueous extracts from plant roots have provided clear evidence for the presence of a rhizoferrin type siderophore, named glomuferrin, in root tissue of mycorrhizal seedlings. Results from HPLC analytical work are seconded by molecular biological data: A BLASTp search revealed that the AM fungal species Gigaspora rosea, Rhizophagus irregularis (formerly Glomus intraradices), Glomus cerebriformis and Diversispora epigea encode a non-ribosomal peptide synthetase (NRPS)-independent siderophore synthase (NIS), which is homologous to the rhizoferrin synthetase of Rhizopus delemar. Thus this study indicates that the biosynthesis of rhizoferrin type siderophores such as glomuferrin (= bis-imidorhizoferrin) may be widespread in the AM symbiosis.

Different Genetic Sources Contribute to the Small RNA Population in the Arbuscular Mycorrhizal Fungus Gigaspora margarita.

RNA interference (RNAi) is a key regulatory pathway of gene expression in almost all eukaryotes. This mechanism relies on short non-coding RNA molecules (sRNAs) to recognize in a sequence-specific manner DNA or RNA targets leading to transcriptional or post-transcriptional gene silencing. To date, the fundamental role of sRNAs in the regulation of development, stress responses, defense against viruses and mobile elements, and cross-kingdom interactions has been extensively studied in a number of biological systems. However, the knowledge of the “RNAi world” in arbuscular mycorrhizal fungi (AMF) is still limited. AMF are obligate mutualistic endosymbionts of plants, able to provide several benefits to their partners, from improved mineral nutrition to stress tolerance. Here we described the RNAi-related genes of the AMF Gigaspora margarita and characterized, through sRNA sequencing, its complex small RNAome, considering the possible genetic sources and targets of the sRNAs. G. margarita indeed is a mosaic of different genomes since it hosts endobacteria, RNA viruses, and non-integrated DNA fragments corresponding to mitovirus sequences. Our findings show that G. margarita is equipped with a complete set of RNAi-related genes characterized by the expansion of the Argonaute-like (AGO-like) gene family that seems a common trait of AMF. With regards to sRNAs, we detected populations of sRNA reads mapping to nuclear, mitochondrial, and viral genomes that share similar features (25-nt long and 5′-end uracil read enrichments), and that clearly differ from sRNAs of endobacterial origin. Furthermore, the annotation of nuclear loci producing sRNAs suggests the occurrence of different sRNA-generating processes. In silico analyses indicate that the most abundant G. margarita sRNAs, including those of viral origin, could target transcripts in the host plant, through a hypothetical cross-kingdom RNAi.

Steel slag and phosphate nutrition of corn inoculated with arbuscular mycorrhizal fungi

Abstract: The objective of this work was to evaluate the effect of the use of steel slag as a soil acidity corrective and of mycorrhizal fungi associated with phosphate fertilization on corn plants. The study was performed in a greenhouse, using 3-kg pots with a Typic Haplorthox, in a 4x5x2 factorial arrangement, with four acidity correction treatments (C1, correction with dolomitic limestone at a dose of 4 Mg ha-1; C2, correction with steel slag at a dose of 4 Mg ha-1; C3, correction with a 1:1 mixture of 2 Mg ha-1 dolomitic limestone and 2 Mg ha-1 steel slag; and C4, control, without pH correction), five phosphorus doses (0, 42, 95, 213, and 480 mg dm-3), and the presence or absence of two arbuscular mycorrhizal fungi (Rhizophagus clarus and Gigaspora margarita), with five replicates. Steel slag was efficient in correcting soil pH and providing Ca and Mg for the plants; therefore, it could replace limestone. The inoculation with R. clarus and G. margarita, associated with P doses of 42, 95, and 213 mg dm-3, improved the development of corn plants after 45 days, resulting in greater plant height, stem diameter, leaf area, and shoot and root dry matter.

Metabolome changes are induced in the arbuscular mycorrhizal fungus Gigaspora margarita by germination and by its bacterial endosymbiont.

Metabolomic profiling is becoming an increasingly important technique in the larger field of systems biology by allowing the simultaneous measurement of thousands of small molecules participating in and resulting from cellular reactions. In this way, metabolomics presents an opportunity to observe the physiological state of a system, which may provide the ability to monitor the whole of cellular metabolism as the technology progresses. The arbuscular mycorrhizal fungus Gigaspora margarita has not previously been explored with regard to metabolite composition. To develop a better understanding of G. margarita and the influences of its endosymbiont Candidatus Glomeribacter gigasporarum, a metabolomic analysis was applied to quiescent and germinated spores with and without endobacteria. Over 100 metabolites were identified and greater than 2600 unique unidentified spectral features were observed. Multivariate analysis of the metabolomes was performed, and a differentiation between all metabolic states of spores and spores hosting the endobacteria was observed. The known metabolites were recruited to many biochemical pathways, with many being involved in maintenance of the antioxidant potential, tyrosine metabolism, and melanin production. Each of the pathways had higher metabolite abundances in the presence of the endosymbiont. These metabolomics data also agree with previously reported transcriptomics results demonstrating the capability of this technique to confirm hypotheses and showing the feasibility of multi-omic approaches for the study of arbuscular mycorrhizal fungi and their endobacterial communities. Challenges still exist in metabolomic analysis, e.g., the identification of compounds is demanding due to incomplete libraries. A metabolomics technique to probe the effects of bacterial endosymbionts on fungal physiology is presented herein, and this method is useful for hypothesis generation as well as testing as noted above.

The virome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals the first report of DNA fragments corresponding to replicating non-retroviral RNA viruses in fungi.

Arbuscular Mycorrhizal Fungi (AMF) are key components of the plant microbiota. AMF genetic complexity is increased by the presence of endobacteria, which live inside many species. A further component of such complexity is the virome associated to AMF, whose knowledge is still very limited. Here, by exploiting transcriptomic data we describe the virome of Gigaspora margarita. A BLAST search for viral RNA-dependent RNA polymerases sequences allowed the identification of four mitoviruses, one Ourmia-like narnavirus, one Giardia-like virus, and two sequences related to Fusarium graminearum mycoviruses. Northern blot and RT-PCR confirmed the authenticity of all the sequences with the exception of the F. graminearum-related ones. All the mitoviruses are replicative and functional since both positive strand and negative strand RNA are present. The abundance of the viral RNA molecules is not regulated by the presence or absence of Candidatus Glomeribacter gigasporarum, the endobacterium hosted by G. margarita, with the exception of the Ourmia-like sequence which is absent in bacteria-cured spores. In addition, we report, for the first time, DNA fragments corresponding to mitovirus sequences associated to the presence of viral RNA. These sequences are not integrated in the mitochondrial DNA and preliminary evidence seems to exclude integration in the nuclear genome.

Strigolactones cross the kingdoms: plants, fungi, and bacteria in the arbuscular mycorrhizal symbiosis.

Strigolactones (SLs) first evolved as regulators of simple developmental processes in very ancient plant lineages, and then assumed new roles to sustain the increasing biological complexity of land plants. Their versatility is also shown by the fact that during evolution they have been exploited, once released in the rhizosphere, as a communication system towards plant-interacting organisms even belonging to different kingdoms. Here, we reviewed the impact of SLs on soil microbes, paying particular attention to arbuscular mycorrhizal fungi (AMF). SLs induce several responses in AMF, including spore germination, hyphal branching, mitochondrial metabolism, transcriptional reprogramming, and production of chitin oligosaccharides which, in turn, stimulate early symbiotic responses in the host plant. In the specific case study of the AMF Gigaspora margarita, SLs are also perceived, directly or indirectly, by the well-characterized population of endobacteria, with an increase of bacterial divisions and the activation of specific transcriptional responses. The dynamics of SLs during AM root colonization were also surveyed. Although not essential for the establishment of this mutualistic association, SLs act as positive regulators as they are relevant to achieve the full extent of colonization. This possibly occurs through a complex crosstalk with other hormones such as auxin, abscisic acid, and gibberellins.

Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in arbuscular mycorrhizal fungi

Hyphal branching in the vicinity of host roots is a host recognition response of arbuscular mycorrhizal fungi. This morphological event is elicited by strigolactones. Strigolactones are carotenoid-derived terpenoids that are synthesized from carlactone and its oxidized derivatives. To test the possibility that carlactone and its oxidized derivatives might act as host-derived precolonization signals in arbuscular mycorrhizal symbiosis, carlactone, carlactonoic acid, and methyl carlactonoate as well as monohydroxycarlactones, 4-, 18-, and 19-hydroxycarlactones, were synthesized chemically and evaluated for hyphal branching-inducing activity in germinating spores of the arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal branching activity was found to correlate with the degree of oxidation at C-19 methyl. Carlactone was only weakly active (100 ng/disc), whereas carlactonoic acid showed comparable activity to the natural canonical strigolactones such as strigol and sorgomol (100 pg/disc). Hydroxylation at either C-4 or C-18 did not significantly affect the activity. A series of carlactone analogues, named AD ester and AA’D diester, was synthesized by reacting formyl Meldrum’s acid with benzyl, cyclohexylmethyl, and cyclogeranyl alcohols (the A-ring part), followed by coupling of the potassium enolates of the resulting formylacetic esters with the D-ring butenolide. AD ester analogues exhibited moderate activity (1 ng–100 pg/disc), while AA’D diester analogues having cyclohexylmethyl and cyclogeranyl groups were highly active on the AM fungus (10 pg/disc). These results indicate that the oxidation of methyl to carboxyl at C-19 in carlactone is a prerequisite but BC-ring formation is not essential to show hyphal branching activity comparable to that of canonical strigolactones.

Crescimento e produção de capítulos florais de calêndula em resposta à inoculação micorrízica e fósforo

RESUMO: O objetivo foi avaliar o efeito da inoculação com fungos micorrízicos arbusculares e fósforo no crescimento e produção de capítulos florais da calêndula (Calendula officinalis). O experimento foi conduzido em casa de vegetação, em vasos. Foram feitas inoculações com os fungos micorrízicos arbusculares Glomus clarum, Gigaspora margarita e controle (sem fungo) e adubadas ou não com fósforo (50 mg/dm3). O arranjo foi em fatorial 3 x 2, no delineamento experimental blocos casualizados, com quatro repetições. Em solos sem adição de adubação fosfatada, plantas de lendula officinalis, inoculadas com o fungo micorrízico arbuscular Gigaspora margarita, apresentaram maior altura (27,9 cm), maior massa seca da parte aérea (4,22 g/planta) e da raiz, maior número (16,5) e maior massa seca de capítulos florais (0,51 g/capítulo) em comparação com plantas não micorrizadas (19,8 cm, 3,49 g/planta, 7,0 e 0,22 g/capítulo, respectivamente).

Mycoavidus cysteinexigens gen. nov., sp. nov., an endohyphal bacterium isolated from a soil isolate of the fungus Mortierella elongata.

An endohyphal bacterium (strain B1-EBT) living in association with the fungus Mortierella elongata FMR23-6 I-B1 was isolated from a fungal cell homogenate and studied for its taxonomic allocation. Cells were Gram-stain-negative, rod-shaped, non-spore-forming, non-motile, and negative for oxidase and catalase. Strain B1-EBT required cysteine for growth and grew at temperatures between 4 and 35 °C. A comparative analysis of 16S rRNA gene sequences revealed that strain B1-EBT forms a distinct clade in the family Burkholderiaceae, encompassing a group of endosymbionts associated with several soil isolates of M. elongata. The most closely related genus is 'Candidatus Glomeribacter gigasporarum', an endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. The major cellular fatty acids of strain B1-EBT were C16 : 0, summed feature 3 (C16 : 1ω7c and C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c or C18 : 1ω6c). Ubiquinone Q-8 was the only quinone detected. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unknown aminophospholipid and two unknown aminolipids. The DNA G+C content was 49.8 mol%. On the basis of phenotypic, chemotaxonomic, and phylogenetic characteristics, strain B1-EBT represents a novel genus and novel species in the family Burkholderiaceae, for which the name Mycoavidus cysteinexigens gen. nov., sp. nov. is proposed. The type strain is B1-EBT ( = JCM 30646T = LMG 28693T = NBRC 110909T).

An interdomain network: the endobacterium of a mycorrhizal fungus promotes antioxidative responses in both fungal and plant hosts

Arbuscular mycorrhizal fungi (AMF) are obligate plant biotrophs that may contain endobacteria in their cytoplasm. Genome sequencing of Candidatus Glomeribacter gigasporarum revealed a reduced genome and dependence on the fungal host. RNA-seq analysis of the AMF Gigaspora margarita in the presence and absence of the endobacterium indicated that endobacteria have an important role in the fungal pre-symbiotic phase by enhancing fungal bioenergetic capacity. To improve the understanding of fungal-endobacterial interactions, iTRAQ (isobaric tags for relative and absolute quantification) quantitative proteomics was used to identify differentially expressed proteins in G.margarita germinating spores with endobacteria (B+), without endobacteria in the cured line (B-) and after application of the synthetic strigolactone GR24. Proteomic, transcriptomic and biochemical data identified several fungal and bacterial proteins involved in interspecies interactions. Endobacteria influenced fungal growth, calcium signalling and metabolism. The greatest effects were on fungal primary metabolism and respiration, which was 50% higher in B+ than in B-. A shift towards pentose phosphate metabolism was detected in B-. Quantification of carbonylated proteins indicated that the B- line had higher oxidative stress levels, which were also observed in two host plants. This study shows that endobacteria generate a complex interdomain network that affects AMF and fungal-plant interactions.

Effect of volatiles versus exudates released by germinating spores of Gigaspora margarita on lateral root formation

Arbuscular mycorrhizal (AM) fungi influence the root system architecture of their hosts; however, the underlying mechanisms have not been fully elucidated. Ectomycorrhizal fungi influence root architecture via volatiles. To determine whether volatiles also play a role in root system changes in response to AM fungi, spores of the AM fungus Gigaspora margarita were inoculated on the same plate as either wild type (WT) Lotus japonicus, the L. japonicus mutant Ljcastor (which lacks the symbiotic cation channel CASTOR, which is required for inducing nuclear calcium spiking, which is necessary for symbiotic partner recognition), or Arabidopsis thaliana, separated by cellophane membranes (fungal exudates experiment), or on different media but with a shared head space (fungal volatiles experiment). Root development was monitored over time. Both germinating spore exudates (GSEs) and geminated-spore-emitted volatile organic compounds (GVCs) significantly promoted lateral root formation (LRF) in WT L. japonicus. LRF in Ljcastor was significantly enhanced in the presence of GVCs. GVCs stimulated LRF in A. thaliana, whereas GSEs showed an inhibitory effect. The expression profile of the genes involved in mycorrhizal establishment and root development were investigated using quantitative reverse transcription-PCR analysis. Only the expression of the LjCCD7 gene, an important component of the strigolactone synthesis pathway, was differentially expressed following exposure to GVCs. We conclude that volatile organic compounds released by the germinating AM fungal spores may stimulate LRF in a symbiosis signaling pathway (SYM)- and host-independent way, whereas GSEs stimulate LRF in a SYM- and host-dependent way.

Medicaol, a strigolactone identified as a putative didehydro-orobanchol isomer, from Medicago truncatula

A major strigolactone produced by the model legume Medicago truncatula (barrel medic) has been tentatively identified as a didehydro-orobanchol isomer. In this study, a putative didehydro-orobanchol isomer was isolated from root exudates collected from barrel medic grown hydroponically under phosphate-starved conditions. The structure and absolute configurations of this strigolactone, named medicaol, were determined by a combination of spectroscopic analysis and spectral comparison with 4-deoxymedicaol which was synthesized using solvolysis and rearrangement of hydroxymethylhexahydroindenone to tetrahydroazulenone as a key step. Medicaol has a seven-membered cycloheptadiene in the A ring instead of a typical six-membered cyclohexene. Medicaol and 4-deoxymedicaol showed activity comparable to their corresponding six-membered A ring relatives, orobanchol and 4-deoxyorobanchol (ent-2′-epi-5-deoxystrigol), in inducing hyphal branching of germinating spores of an arbuscular mycorrhizal fungus Gigaspora margarita. Plausible biosynthetic pathways from 4-deoxyorobanchol to medicaol are also proposed.

PH measurement of tubular vacuoles of an arbuscular mycorrhizal fungus, Gigaspora margarita.

Arbuscular mycorrhizal fungi play an important role in phosphate supply to the host plants. The fungal hyphae contain tubular vacuoles where phosphate compounds such as polyphosphate are accumulated. Despite their importance for the phosphate storage, little is known about the physiological properties of the tubular vacuoles in arbuscular mycorrhizal fungi. As an indicator of the physiological state in vacuoles, we measured pH of tubular vacuoles in living hyphae of arbuscular mycorrhizal fungus Gigaspora margarita using ratio image analysis with pH-dependent fluorescent probe, 6-carboxyfluorescein. Fluorescent images of the fine tubular vacuoles were obtained using a laser scanning confocal microscope, which enabled calculation of vacuolar pH with high spatial resolution. The tubular vacuoles showed mean pH of 5.6 and a pH range of 5.1-6.3. These results suggest that the tubular vacuoles of arbuscular mycorrhizal fungi have a mildly acidic pH just like vacuoles of other fungal species including yeast and ectomycorrhizal fungi.

Automated analysis of calcium spiking profiles with CaSA software: two case studies from root-microbe symbioses.

BackgroundRepeated oscillations in intracellular calcium (Ca2+) concentration, known as Ca2+ spiking signals, have been described in plants for a limited number of cellular responses to biotic or abiotic stimuli and most notably the common symbiotic signaling pathway (CSSP) which mediates the recognition by their plant hosts of two endosymbiotic microbes, arbuscular mycorrhizal (AM) fungi and nitrogen fixing rhizobia. The detailed analysis of the complexity and variability of the Ca2+ spiking patterns which have been revealed in recent studies requires both extensive datasets and sophisticated statistical tools.ResultsAs a contribution, we have developed automated Ca2+ spiking analysis (CaSA) software that performs i) automated peak detection, ii) statistical analyses based on the detected peaks, iii) autocorrelation analysis of peak-to-peak intervals to highlight major traits in the spiking pattern.We have evaluated CaSA in two experimental studies. In the first, CaSA highlighted unpredicted differences in the spiking patterns induced in Medicago truncatula root epidermal cells by exudates of the AM fungus Gigaspora margarita as a function of the phosphate concentration in the growth medium of both host and fungus. In the second study we compared the spiking patterns triggered by either AM fungal or rhizobial symbiotic signals. CaSA revealed the existence of different patterns in signal periodicity, which are thought to contribute to the so-called Ca2+ signature.ConclusionsWe therefore propose CaSA as a useful tool for characterizing oscillatory biological phenomena such as Ca2+ spiking.

Interações entre fungos micorrízicos arbusculares, rizóbio e actinomicetos na rizosfera de soja

Isolados de actinomicetos e de fungos micorrízicos arbusculares foram avaliados em sistemas de inoculação conjunta in vivo, em plantas de soja inoculadas com rizóbios, com a finalidade de se observar interações microbianas. A pesquisa foi realizada no Centro Nacional de Pesquisa de Agrobiologia (Embrapa CNPAB). Foram utilizados, como substrato, solo da série Itaguaí (textura média) misturado com areia de rio na proporção 1:1 (v:v), com adição de isolados de actinomicetos (ACT-78 e ACT-370); estirpes de rizóbios (Bradyrhizobium elkanii - BR 29 e Bradyrhizobium japonicum - BR 33) e fungos micorrízicos arbusculares (Gigaspora margarita e Glomus clarum). Os resultados revelam que a inoculação com os fungos micorrízicos arbusculares influenciou de modo expressivo as variáveis de crescimento, nodulação e densidade de actinomicetos na rizosfera de soja, que apresentaram valores maiores do que as plantas sem o inóculo fúngico. Houve efeitos sinergísticos e antagônicos dos fungos micorrízicos arbusculares com os actinomicetos e com rizóbios.

Effects of arbuscular mycorrhizal fungi on the abundance of foliar-feeding insects and their natural enemy

We investigated the effects of symbiotic association between a plant and an arbuscular mycorrhizal fungus (AMF) on the abundance of aboveground foliar-feeding insects that differed in feeding mode and their predator. We examined effects on insect abundance as the result of AMF-related changes in the quality and quantity of plants in the field. The numbers of three insects with different foliar-feeding mode (phloem feeder, chewer, and cell-content feeder) and their generalist predator Orius sauteri Poppius on soybean Glycine max (L.) Merrill with and without the AMF Gigaspora margarita Becker & Hall were compared over time. Symbiotic association between the AMF and the soybean increased shoot biomass, the concentration of phosphorus in the soybean, and the abundance of the phloem feeder Aulacorthum solani Kaltenbach, but did not affect the abundance of generalist chewers. In addition, the effects of the symbiotic association on the abundance of cell-content feeding Thrips spp. and the generalist predator O. sauteri differed between sample dates. These results indicated that the effects of the symbiotic association on the number of foliar-feeding insects depended on feeding mode and the number of predators.

Leaf herbivory by Spodoptera litura increases arbuscular mycorrhizal colonization in roots of soybean

The improvement of host plant growth resulting from mycorrhizal colonization often affects the viability of above-ground herbivorous insects. Conversely, the effects of herbivorous insects on plant growth may indirectly affect below-ground microorganisms. This study investigated the effects of herbivorous insects on arbuscular mycorrhizal (AM) colonization of soybean (Glycine max Merr.). Seeds of soybean were sown in soil inoculated with the AM fungus Gigaspora margarita Becker & Hall and grown in growth chambers. At 39 days after inoculation (DAI), a larva of the common cutworm (Spodoptera litura Fabricius) was placed on a leaf of each soybean plant for 5 days. AM colonization of soybean on 39 DAI was 21%. Although not evident by 62 DAI, by 77 DAI there was a statistically significant increase in AM colonization in plants subjected to herbivory by S. litura relative to control plants. Leaf removal at 46 DAI was also associated with increased AM colonization at 78 DAI. These results suggest that herbivory by chewing insect affects AM colonization and that the effect is likely caused by a decrease in leaf area.

The mitochondrial genome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals two unsuspected trans-splicing events of group I introns.

• Arbuscular mycorrhizal fungi (AMF) are ubiquitous organisms that benefit ecosystems through the establishment of an association with the roots of most plants: the mycorrhizal symbiosis. Despite their ecological importance, however, these fungi have been poorly studied at the genome level. • In this study, total DNA from the AMF Gigaspora margarita was subjected to a combination of 454 and Illumina sequencing, and the resulting reads were used to assemble its mitochondrial genome de novo. This genome was annotated and compared with those of other relatives to better comprehend the evolution of the AMF lineage. • The mitochondrial genome of G. margarita is unique in many ways, exhibiting a large size (97 kbp) and elevated GC content (45%). This genome also harbors molecular events that were previously unknown to occur in fungal mitochondrial genomes, including trans-splicing of group I introns from two different genes coding for the first subunit of the cytochrome oxidase and for the small subunit of the rRNA. • This study reports the second published genome from an AMF organelle, resulting in relevant DNA sequence information from this poorly studied fungal group, and providing new insights into the frequency, origin and evolution of trans-spliced group I introns found across the mitochondrial genomes of distantly related organisms.

The genome of the obligate endobacterium of an AM fungus reveals an interphylum network of nutritional interactions.

As obligate symbionts of most land plants, arbuscular mycorrhizal fungi (AMF) have a crucial role in ecosystems, but to date, in the absence of genomic data, their adaptive biology remains elusive. In addition, endobacteria are found in their cytoplasm, the role of which is unknown. In order to investigate the function of the Gram-negative Candidatus Glomeribacter gigasporarum, an endobacterium of the AMF Gigaspora margarita, we sequenced its genome, leading to an ∼1.72-Mb assembly. Phylogenetic analyses placed Ca. G. gigasporarum in the Burkholderiaceae whereas metabolic network analyses clustered it with insect endobacteria. This positioning of Ca. G. gigasporarum among different bacterial classes reveals that it has undergone convergent evolution to adapt itself to intracellular lifestyle. The genome annotation of this mycorrhizal-fungal endobacterium has revealed an unexpected genetic mosaic where typical determinants of symbiotic, pathogenic and free-living bacteria are integrated in a reduced genome. Ca. G. gigasporarum is an aerobic microbe that depends on its host for carbon, phosphorus and nitrogen supply; it also expresses type II and type III secretion systems and synthesizes vitamin B12, antibiotics- and toxin-resistance molecules, which may contribute to the fungal host's ecological fitness. Ca. G. gigasporarum has an extreme dependence on its host for nutrients and energy, whereas the fungal host is itself an obligate biotroph that relies on a photosynthetic plant. Our work represents the first step towards unraveling a complex network of interphylum interactions, which is expected to have a previously unrecognized ecological impact.

Lupin pyranoisoflavones inhibiting hyphal development in arbuscular mycorrhizal fungi

White lupin ( Lupinus albus L.), a non-host plant for arbuscular mycorrhizal (AM) fungi in the typically mycotrophic family Fabaceae, has been investigated for root metabolites that inhibit hyphal development in AM fungi. Four known pyranoisoflavones, licoisoflavone B ( 1), sophoraisoflavone A ( 2), alpinumisoflavone ( 3) and 3′-hydroxy-4′- O-methylalpinumisoflavone ( 4), together with three previously unknown pyranoisoflavones, lupindipyranoisoflavone A ( 5), 10′-hydroxylicoisoflavone B ( 6) and 10′-hydroxysophoraisoflavone A ( 7) were isolated from the root exudates of white lupin as an inhibitor of germ tube growth in the AM fungus Gigaspora margarita. Pyranoisoflavones 1, 2 and 3 strongly inhibited germ tube growth at 0.63, 1.25 and 0.63 μg/disc, respectively. The remaining compounds 4– 7 were either moderate or weak inhibitors that inhibited germ tube growth at concentrations higher than 10 μg/disc. Licoisoflavone B ( 1) and sophoraisoflavone A ( 2) completely inhibited hyphal branching induced by a lupin strigolactone, orobanchyl acetate, in G. margarita at 0.16 and 0.63 μg/disc, respectively.