Effects of fungal endophytes and arbuscular mycorrhizal fungi on growth of Echium vulgare and alkannin/shikonin and their derivatives production in roots.

Endophytic fungi as well as arbuscular mycorrhizal fungi (AMF) are known to stimulate plant growth and production of secondary metabolites in medicinal plants. Here, 10 endophytic fungi isolated from roots of wild Alkanna tinctoria plants and 5 AMF purchased from the Glomeromycota in vitro collection were evaluated, during two successive three-month greenhouse experiments, on the growth of Echium vulgare and alkannin/shikonin and their derivatives (A/Sd) production in the roots. Some of the endophytic fungi tested significantly increased plant growth parameters as compared to the control: Cladosporium allicinum, Cadophora sp., Clonostachys sp., Trichoderma hispanicum and Leptosphaeria ladina increased root volume, Plectosphaerella sp. And T. hispanicum root fresh weight and root water retention and T. hispanicum plant water retention. However, none of these fungi impacted A/Sd production. Conversely, none of the AMF strains tested impacted plant growth parameters, but those inoculated with Rhizophagus intraradices MUCL 49410 had a significantly higher concentration of alkannin/shikonin (A/S), acetyl-A/S, ß,ß- dimethylacryl-A/S, isovaleryl-A/S and total A/Sd, compared to the control plants. Further studies are needed to investigate the mechanisms involved in the production of A/Sd in plants associated to specific endophytic fungi/AMF and on the cultivation conditions required for optimal production of these compounds.

Arbuscular mycorrhizal fungi impact the production of alkannin/shikonin and their derivatives in Alkanna tinctoria Tausch. grown in semi-hydroponic and pot cultivation systems.

Introduction: Alkanna tinctoria Tausch. is a medicinal plant well-known to produce important therapeutic compounds, such as alkannin/shikonin and their derivatives (A/Sd). It associates with arbuscular mycorrhizal fungi (AMF), which are known, amongst others beneficial effects, to modulate the plant secondary metabolites (SMs) biosynthesis. However, to the best of our knowledge, no study on the effects of AMF strains on the growth and production of A/Sd in A. tinctoria has been reported in the literature. Methods: Here, three experiments were conducted. In Experiment 1, plants were associated with the GINCO strain Rhizophagus irregularis MUCL 41833 and, in Experiment 2, with two strains of GINCO (R. irregularis MUCL 41833 and Rhizophagus aggregatus MUCL 49408) and two native strains isolated from wild growing A. tinctoria (R. irregularis and Septoglomus viscosum) and were grown in a semi-hydroponic (S-H) cultivation system. Plants were harvested after 9 and 37 days in Experiment 1 and 9 days in Experiment 2. In Experiment 3, plants were associated with the two native AMF strains and with R. irregularis MUCL 41833 and were grown for 85 days in pots under greenhouse conditions. Quantification and identification of A/Sd were performed by HPLC-PDA and by HPLC-HRMS/MS, respectively. LePGT1, LePGT2, and GHQH genes involved in the A/Sd biosynthesis were analyzed through RT-qPCR. Results: In Experiment 1, no significant differences were noticed in the production of A/Sd. Conversely, in Experiments 2 and 3, plants associated with the native AMF R. irregularis had the highest content of total A/Sd expressed as shikonin equivalent. In Experiment 1, a significantly higher relative expression of both LePGT1 and LePGT2 was observed in plants inoculated with R. irregularis MUCL 41833 compared with control plants after 37 days in the S-H cultivation system. Similarly, a significantly higher relative expression of LePGT2 in plants inoculated with R. irregularis MUCL 41833 was noticed after 9 versus 37 days in the S-H cultivation system. In Experiment 2, a significant lower relative expression of LePGT2 was observed in native AMF R. irregularis inoculated plants compared to the control. Discussion: Overall, our study showed that the native R. irregularis strain increased A/Sd production in A. tinctoria regardless of the growing system used, further suggesting that the inoculation of native/best performing AMF is a promising method to improve the production of important SMs.

Hyphosphere interactions between Rhizophagus irregularis and Rahnella aquatilis promote carbon-phosphorus exchange at the peri-arbuscular space in Medicago truncatula.

Arbuscular mycorrhizal (AM) fungi form a continuum between roots and soil. One end of this continuum is comprised of the highly intimate plant-fungus interface with intracellular organelles for nutrient exchange, while on the other end the fungus interacts with bacteria to compensate for the AM fungus' inability to take up organic nutrients from soil. How both interfaces communicate in this highly complex tripartite mutualism is widely unknown. Here, the effects of phosphate-solubilizing bacteria (PSB) Rahnella aquatilis dwelling at the surface of the extraradical hyphae of Rhizophagus irregularis was analysed based on the expression of genes involved in C-P exchange at the peri-arbuscular space (PAS) in Medicago truncatula. The interaction between AM fungus and PSB resulted in an increase in uptake and transport of Pi along the extraradical hyphae and its transfer from AM fungus to plant. In return, this was remunerated by a transfer of C from plant to AM fungus, improving the C-P exchange at the PAS. These results demonstrated that a microorganism (i.e., a PSB) developing at the hyphosphere interface can affect the C-P exchange at the PAS between plant and AM fungus, suggesting a fine-tuned communication operated between three organisms via two distantly connected interfaces.

Root-associated bacteria modulate the specialised metabolome of Lithospermum officinale L.

Bacteria influence plant growth and development and therefore are attractive resources for applications in agriculture. However, little is known about the impact of these microorganisms on secondary metabolite (SM) production by medicinal plants. Here we assessed, for the first time, the effects of bacteria on the modulation of SM production in the medicinal plant Lithospermum officinale (Boraginaceae family) with a focus on the naphthoquinones alkannin/shikonin and their derivatives (A/Sd). The study was conducted in an in vitro cultivation system developed for that purpose, as well as in a greenhouse. Targeted and non-targeted metabolomics were performed, and expression of the gene PGT encoding for a key enzyme in the A/S biosynthesis pathway was evaluated with qPCR. Three strains, Chitinophaga sp. R-73072, Xanthomonas sp. R-73098 and Pseudomonas sp. R-71838 induced a significant increase of A/Sd in L. officinale in both systems, demonstrating the strength of our approach for screening A/Sd-inducing bacteria. The bacterial treatments altered other plant metabolites derived from the shikimate pathway as well. Our results demonstrate that bacteria influence the biosynthesis of A/Sd and interact with different metabolic pathways. This work highlights the potential of bacteria to increase the production of SM in medicinal plants and reveals new patterns in the metabolome regulation of L. officinale.

Metabolite Production in Alkanna tinctoria Links Plant Development with the Recruitment of Individual Members of Microbiome Thriving at the Root-Soil Interface.

Plants are naturally associated with diverse microbial communities, which play significant roles in plant performance, such as growth promotion or fending off pathogens. The roots of Alkanna tinctoria L. are rich in naphthoquinones, particularly the medicinally used enantiomers alkannin and shikonin and their derivatives. Former studies already have shown that microorganisms may modulate plant metabolism. To further investigate the potential interaction between A. tinctoria and associated microorganisms, we performed a greenhouse experiment in which A. tinctoria plants were grown in the presence of three distinct soil microbiomes. At four defined plant developmental stages, we made an in-depth assessment of bacterial and fungal root-associated microbiomes as well as all extracted primary and secondary metabolite content of root material. Our results showed that the plant developmental stage was the most important driver influencing the plant metabolite content, revealing peak contents of alkannin/shikonin derivatives at the fruiting stage. Plant root microbial diversity was influenced both by bulk soil origin and to a small extent by the developmental stage. The performed correlation analyses and cooccurrence networks on the measured metabolite content and the abundance of individual bacterial and fungal taxa suggested a dynamic and at times positive or negative relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Labrys and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium as well as four species of the fungal genus Penicillium were found to be positively correlated with higher content of alkannins. IMPORTANCE Previous studies have shown that individual, isolated microorganisms may influence secondary metabolism of plants and induce or stimulate the production of medicinally relevant secondary metabolism. Here, we analyzed the microbiome-metabolome linkage of the medicinal plant Alkanna tinctoria, which is known to produce valuable compounds, particularly the naphthoquinones alkannin and shikonin and their derivatives. A detailed bacterial and fungal microbiome and metabolome analysis of A. tinctoria roots revealed that the plant developmental stage influenced root metabolite production, whereas soil inoculants from three different geographical origins in which plants were grown shaped root-associated microbiota. Metabolomes of plant roots of the same developmental stage across different soils were highly similar, pinpointing to plant maturity as the primary driver of secondary metabolite production. Correlation and network analyses identified bacterial and fungal taxa showing a positive relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Labrys as well as the fungal species of genus Penicillium were found to be positively correlated with higher content of alkannins.

Arbuscular mycorrhizal fungi and production of secondary metabolites in medicinal plants.

Medicinal plants are an important source of therapeutic compounds used in the treatment of many diseases since ancient times. Interestingly, they form associations with numerous microorganisms developing as endophytes or symbionts in different parts of the plants. Within the soil, arbuscular mycorrhizal fungi (AMF) are the most prevalent symbiotic microorganisms forming associations with more than 70% of vascular plants. In the last decade, a number of studies have reported the positive effects of AMF on improving the production and accumulation of important active compounds in medicinal plants.In this work, we reviewed the literature on the effects of AMF on the production of secondary metabolites in medicinal plants. The major findings are as follows: AMF impact the production of secondary metabolites either directly by increasing plant biomass or indirectly by stimulating secondary metabolite biosynthetic pathways. The magnitude of the impact differs depending on the plant genotype, the AMF strain, and the environmental context (e.g., light, time of harvesting). Different methods of cultivation are used for the production of secondary metabolites by medicinal plants (e.g., greenhouse, aeroponics, hydroponics, in vitro and hairy root cultures) which also are compatible with AMF. In conclusion, the inoculation of medicinal plants with AMF is a real avenue for increasing the quantity and quality of secondary metabolites of pharmacological, medical, and cosmetic interest.

Two-component system in Rahnella aquatilis is impacted by the hyphosphere of the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Two-component systems (TCS) are ubiquitous among bacteria, playing key roles in signalling events. However, to what extent the TCS of Rahnella aquatilis (a Phosphate solubilizing bacteria) is influenced by the hyphosphere of the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis is totally unknown. Here, the expression of 16 genes encoding the TCS of R. aquatilis (i.e. involved in carbon-sensing and nutrient-sensing) and of eight genes regulated by the PhoR TCS (i.e. involved in inorganic and organic phosphorus mobilization) were analysed at regular intervals in presence of hyphae of R. irregularis. The study was conducted under in vitro culture conditions with phytate as the unique source of phosphorus. In presence of the AM fungus, the expression of TCS genes involved in carbon-sensing and nutrient-sensing were stimulated. Only, BaeS at 30 and 120 min, and BaeR at 60 min were inhibited. In addition, the PhoR TCS stimulated the expression of genes encoding phosphatase but inhibited the expression of genes involved in gluconic acid production. As the mechanism of coupling environmental changes with cellular physiological changes, TCS plays a pivotal role in regulating specific gene expression in R. aquatilis, recognizing environmental signals. More importantly, TCS genes may regulate bacteria response to hyphal carbon to mobilize phosphorus efficiently in the hyphosphere.

Genetic analysis of tomato root colonization by arbuscular mycorrhizal fungi.

BACKGROUND AND AIMS: Arbuscular mycorrhizal fungi (AMF) play an important role in plant nutrition and protection against pests and diseases, as well as in soil structuration, nutrient cycling and, generally speaking, in sustainable agriculture, particularly under drought, salinity and low input or organic agriculture. However, little is known about the genetics of the AMF-plant association in tomato. The aim of this study was the genetic analysis of root AMF colonization in tomato via the detection of the quantitative trait loci (QTLs) involved. METHODS: A population of 130 recombinant inbred lines derived from the wild species Solanum pimpinellifolium, genotyped for 1899 segregating, non-redundant single nucleotide polymorphisms (SNPs) from the SolCAP tomato panel, was characterized for intensity, frequency and arbuscular abundance of AMF colonization to detect the QTLs involved and to analyse the genes within their peaks (2-2.6 Mbp). KEY RESULTS: The three AMF colonization parameters were highly correlated (0.78-0.97) and the best one, with the highest heritability (0.23), corresponded to colonization intensity. A total of eight QTLs in chromosomes 1, 3, 4, 5, 6, 8, 9 and 10 were detected. Seven of them simultaneously affected intensity and arbuscule abundance. The allele increasing the expression of the trait usually came from the wild parent in accordance with the parental means, and several epistatic interactions were found relevant for breeding purposes. SlCCaMK and SlLYK13 were found among the candidate genes. Carbohydrate transmembrane transporter activity, lipid metabolism and transport, metabolic processes related to nitrogen and phosphate-containing compounds, regulation of carbohydrates, and other biological processes involved in the plant defence were found to be over-represented within the QTL peaks. CONCLUSIONS: Intensity is genetically the best morphological measure of tomato root AMF colonization. Wild alleles can improve AMF colonization, and the gene contents of AMF colonization QTLs might be important for explaining the establishment and functioning of the AMF-plant symbiosis.

Community composition of arbuscular mycorrhizal fungi associated with native plants growing in a petroleum-polluted soil of the Amazon region of Ecuador.

Arbuscular mycorrhizal fungi (AMF) are worldwide distributed plant symbionts. However, their occurrence in hydrocarbon-polluted environments is less investigated, although specific communities may be present with possible interest for remediation strategies. Here, we investigated the AMF community composition associated with the roots of diverse plant species naturally recolonizing a weathered crude oil pond in the Amazon region of Ecuador. Next generation 454 GS-Junior sequencing of an 800 bp LSU rRNA gene PCR amplicon was used. PCR amplicons were affiliated to a maximum-likelihood phylogenetic tree computed from 1.5 kb AMF reference sequences. A high throughput phylogenetic annotation approach, using an evolutionary placement algorithm (EPA) allowed the characterization of sequences to the species level. Fifteen species were detected. Acaulospora species were identified as dominant colonizers, with 73% of relative read abundance, Archaeospora (19.6%) and several genera from the Glomeraceae (Rhizophagus, Glomus macrocarpum-like, Sclerocystis, Dominikia and Kamienskia) were also detected. Although, a diverse community belonging to Glomeraceae was revealed, they represented <10% of the relative abundance in the Pond. Seventy five % of the species could not be identified, suggesting possible new species associated with roots of plants under highly hydrocarbon-polluted conditions.

The arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 increases the phosphorus uptake and biomass of Medicago truncatula, a benzo[a]pyrene-tolerant plant species.

The accumulation of phosphorus (P) in plants increases their biomass and resistance/tolerance to organic pollutants. Both characteristics are mandatory for the utilization of plants in phytoremediation. Arbuscular mycorrhizal (AM) fungi improve plant P nutrition, and thus growth. However, only a few studies have focused on the dynamics of inorganic P (Pi) uptake in AM fungal-colonized plants in the presence of organic pollutants. Indeed, most of the results so far were obtained after harvesting the plants, thus by evaluating P concentration and content at a single time point. Here, we investigated the effects of the AM fungus Rhizophagus irregularis MUCL 41833 on the short-term Pi uptake dynamics of Medicago truncatula plants grown in the presence of benzo[a]pyrene (B[a]P), a polyaromatic hydrocarbon (PAH) frequently found in polluted soils. The study was conducted using a non-destructive circulatory semi-hydroponic cultivation system to investigate the short-term Pi depletion from a nutrient solution and as a corollary, the Pi uptake by the AM fungal-colonized and non-colonized plants. The growth, P concentration, and content of plants were also evaluated at harvest. The presence of B[a]P neither impacted the development of the AM fungus in the roots nor the plant growth and Pi uptake, suggesting a marked tolerance of both organisms to B[a]P pollution. A generally higher Pi uptake coupled with a higher accumulation of P in shoots and roots was noticed in AM fungal-colonized plants as compared to the non-colonized controls, irrespective of the presence or absence of B[a]P. Therefore, fungal-colonized plants showed the best growth. Furthermore, the beneficial effect provided by the presence of the AM fungus in roots was similar in presence or absence of B[a]P, thus opening the door for potential utilization in phytomanagement of PAH-polluted soils.

Increased Silicon Acquisition in Bananas Colonized by Rhizophagus irregularis MUCL 41833 Reduces the Incidence of Pseudocercospora fijiensis.

This work aimed to test the hypothesis that the combination of arbuscular mycorrhizal fungi (AMF) and accumulation of silicon (Si) in banana plants via its uptake and transport by the fungus reduces the incidence of Black Leaf Steak Disease (BLSD) caused by Pseudocercospora fijiensis. Methods: A pot experiment was conducted to compare BLSD symptoms on leaves of banana plants colonized or not by the AMF Rhizophagus irregularis MUCL 41833 and exposed or not to Si added to the growth substrate. Results: A marked increase in plant growth parameters (i.e., pseudostem diameter and height, leaf surface area, shoot, root and total dry weight) as well as accumulation of Si, P, and Ca were noticed in the AMF-colonized banana plants in presence as well as in absence of Si added to the growth substrate. Similarly Si addition to the substrate increased plant growth parameters. Leave symptoms caused by the pathogen were observed in all the treatments but were reduced in presence of AMF as well as in presence of Si added to the growth substrate. The more drastic reduction was noticed in the AMF-colonized plants with Si added to the growth substrate. The Severity Index as well as Area Under Disease Progress Curve were considerably decreased both at 21 (∼48% and 48%, respectively) and 35 days (∼21% and ∼32%, respectively) after inoculation of the pathogen as compared with non-AMF-colonized plants in absence of Si added to the substrate. Conclusion: Our findings revealed that AMF-colonized banana plants grown in a subs-trate supplemented with Si were less impacted by P. fijiensis than non-colonized plants grown without Si added to the growth substrate. The combination of AMF-colonized banana plants (during the weaning phase or in vitro) with the application of Si to soil seems thus a thoughtful option to mitigate the impact of BLSD in bananas, although such strategy needs first to be evaluated under field conditions to appraise its real potential.

Dynamics of Short-Term Phosphorus Uptake by Intact Mycorrhizal and Non-mycorrhizal Maize Plants Grown in a Circulatory Semi-Hydroponic Cultivation System.

A non-destructive cultivation system was developed to study the dynamics of phosphorus (Pi) uptake by mycorrhizal and non-mycorrhizal maize plantlets. The system consisted of a plant container connected via silicon tubes to a glass bottle containing a nutrient solution supplemented with Pi. The nutrient solution is pumped with a peristaltic pump to the upper part of the container via the silicon tubes and the solution percolate through the plantlet container back into the glass bottle. Pi is sampled from the glass bottle at regular intervals and concentration evaluated. Maize plantlets were colonized by the AMF Rhizophagus irregularis MUCL 41833 and Pi uptake quantified at fixed intervals (9, 21, and 42 h) from the depletion of the Pi in the nutrient solution flowing through the plantlets containers. Plants and fungus grew well in the perlite substrate. The concentration of Pi in the bottles followed an almost linear decrease over time, demonstrating a depletion of Pi in the circulating solution and a concomitant uptake/immobilization by the plantlet-AMF associates in the containers. The Pi uptake rate was significantly increased in the AMF-colonized plantlets (at 9 and 21 h) as compared to non-colonized plantlets, although no correlation was noticed with plant growth or P accumulation in shoots. The circulatory semi-hydroponic cultivation system developed was adequate for measuring Pi depletion in a nutrient solution and by corollary Pi uptake/immobilization by the plant-AMF associates. The measurements were non-destructive so that the time course of Pi uptake could be monitored without disturbing the growth of the plant and its fungal associate. The system further opens the door to study the dynamics of other micro and macro-nutrients as well as their uptake under stressed growth conditions such as salinity, pollution by hydrocarbon contaminants or potential toxic elements.

Short-term chromium (VI) exposure increases phosphorus uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833.

Hexavalent chromium is a potent carcinogen, while phosphorus is an essential nutrient. The role of arbuscular mycorrhizal fungi (AMF) in the uptake of P is well known and was also reported, at low levels, for Cr. However, it is unclear whether the uptake of Cr can impact the short-term uptake dynamics of P since both elements have a similar chemical structure and may thus potentially compete with each other during the uptake process. This study investigated the impact of Cr(VI) on short-term P uptake by the AMF Rhizophagus irregularis MUCL 41833 in Medicago truncatula. Bi-compartmented Petri plates were used to spatially separate a root compartment (RC) from a hyphal compartment (HC) using a whole plant in vitro culture system. The HC was supplemented with Cr(VI). Chromium(VI) as well as total Cr and P were monitored during 16 h within the HC and their concentrations determined by the end of the experiment within roots and shoots. Our results indicated that the uptake and translocation of Cr from hyphae to roots was a fast process: roots in which the extraradical mycelium (ERM) was exposed to Cr(VI) accumulated more Cr than roots of which the ERM was not exposed to Cr(VI) or was dead. Our results further confirmed that dead ERM immobilized more Cr than alive ERM. Finally our results demonstrated that the short exposure to Cr(VI) was sufficient to stimulate P uptake by the ERM and that the stimulation process began within the first 4 h of exposure.

Rhizosphere microbiomes of potato cultivated in the High Andes show stable and dynamic core microbiomes with different responses to plant development.

The rhizosphere hosts a rich microflora supporting plant nutrition and health. We examined bacterial rhizosphere microbiota of Solanum tuberosum grown in its center of origin, the Central Andean Highlands, at different vegetation stages and sites at altitudes ranging from 3245 to 4070 m.a.s.l., differing in soil characteristics, climate and the agricultural practices by 454 sequence analysis of 16S rRNA genes. We observed that the taxonomic composition of bacteria repeatedly occurring at particular stages of plant development was almost unaffected by highly diverse environmental conditions. A detailed statistical analysis on the operational taxonomic unit (OTU) level, representing bacterial species, revealed a complex community structure of the rhizosphere. We identified an opportunistic microbiome which comprises OTUs that occur randomly or under specific environmental conditions. In contrast, core microbiome members were found at all sites. The 'stable' component of the core microbiome consisted of few ubiquitous OTUs that were continuously abundant in all samples and vegetation stages, whereas the 'dynamic' component comprised OTUs that were enriched at specific vegetation stages.

Increasing phosphorus concentration in the extraradical hyphae of Rhizophagus irregularis DAOM 197198 leads to a concomitant increase in metal minerals.

Plants associated with arbuscular mycorrhizal fungi (AMF) acquire phosphorus via roots and extraradical hyphae. How soil P level affects P accumulation within hyphae and how P in hyphae influences the accumulation of metal minerals remains little explored. A bi-compartmented in vitro cultivation system separating a root compartment (RC), containing a Ri T-DNA transformed carrot root associated to the AMF Rhizophagus irregularis DAOM 197198, from a hyphal compartment (HC), containing only the extraradical hyphae, was used. The HC contained a liquid growth medium (i.e., the modified Strullu-Romand medium containing P in the form of KH2PO4) without (0 µM) or adjusted to 35, 100, and 700 µM of KH2PO4. The accumulation of P and metal minerals (Ca, Mg, K, Na, Fe, Cu, Mn) within extraradical hyphae and AMF-colonized roots, and the expression of the phosphate transporter gene GintPT were assessed. The expression of GintPT in the extraradical hyphae did not differ in absence of KH2PO4 or in presence of 35 and 100 µM KH2PO4 in the HC but was markedly reduced in presence of 700 µM KH2PO4. Hyphal P concentration was significantly lowest in absence of KH2PO4, intermediate at 35 and 100 µM KH2PO4 and significantly highest in presence of 700 µM KH2PO4 in the HC. The concentrations of K, Mg, and Na were positively associated with the concentration of P in the extraradical hyphae developing in the HC. Similarly, P concentration in extraradical hyphae in the HC was related to P concentration in the growth medium and influenced the concentration of K, Mg, and Na. The accumulation of the metal mineral K, Mg, and Na in the extraradical hyphae developing in the HC was possibly related to their function in neutralizing the negative charges of PolyP accumulated in the hyphae.

Rhizophagus irregularis MUCL 41833 can colonize and improve P uptake of Plantago lanceolata after exposure to ionizing gamma radiation in root organ culture.

Long-lived radionuclides such as (90)Sr and (137)Cs can be naturally or accidentally deposited in the upper soil layers where they emit ß/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 (137)Cs 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.

The induction of Ethylene response factor 3 (ERF3) in potato as a result of co-inoculation with Pseudomonas sp. R41805 and Rhizophagus irregularis MUCL 41833 - a possible role in plant defense.

Colonization of plant rhizosphere/roots by beneficial microorganisms (e.g. plant growth promoting rhizobacteria - PGPR, arbuscular mycorrhizal fungi - AMF) confers broad-spectrum resistance to virulent pathogens and is known as induced systemic resistance (ISR) and mycorrhizal-induced resistance (MIR). ISR or MIR, an indirect mechanism for biocontrol, involves complex signaling networks that are regulated by several plant hormones, the most important of which are salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). In the present study, we investigated if inoculation of potato plantlets with an AMF (Rhizophagus irregularis MUCL 41833) and a PGPR (Pseudomonas sp R41805) either alone or in combination, could elicit host defense response genes in the presence or absence of Rhizoctonia Solani EC-1, a major potato pathogen. RT-qPCR revealed the significant expression of ethylene response factor 3 (EFR3) in mycorrhized potato plantlets inoculated with Pseudomonas sp R41805 and also in mycorrhized potato plantlets inoculated with Pseudomonas sp R41805 and challenged with R. solani. The significance of ethylene response factors (ERFs) in pathogen defense has been well documented in the literature. The results of the present study suggest that the dual inoculation of potato with PGPR and AMF may play a part in the activation of plant systemic defense systems via ERF3.

Polyaromatic hydrocarbons impair phosphorus transport by the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Phosphate uptake by plant roots is mainly mediated by arbuscular mycorrhizal fungi (AMF). However, the impact on phosphorus (P) transport of polycyclic aromatic hydrocarbons (PAH), persistent organic pollutants widely found in altered soils, is not known up today. Here, we monitored the Rhizophagus irregularis fungal growth and the fungal P transport ability from the extraradical mycelium to the host transformed chicory roots in the presence of anthracene and benzo[a]pyrene (B[a]P) and the combination of both PAH, under in vitro conditions. Firstly, our findings showed that PAH have detrimental effect on the fungal growth. The combination of both PAH was more toxic than each of the PAH individually due to synergistic effects. Secondly, PAH affected the P transport by the fungus from the medium to the roots. This was evidenced by either the decrease in (33)P quantity transported in the roots as well as the decrease in acid phosphatase activity in the mycorrhizal roots. Moreover, the fungal alkaline phosphatase activities remained constant in the extraradical mycelium as well as in the roots in the absence and in the presence of PAH. The GintPT and GiALP (encoding a P transporter and an alkaline phosphatase respectively) gene expressions were also found to be similar in the extraradical mycelium treated with PAH or not (control). These findings suggested that the P uptake by R. irregularis was not affected by PAH but probably the transport from the extraradical mycelium to the intraradical mycelium.

Study in vitro of the impact of endophytic bacteria isolated from Centella asiatica on the disease incidence caused by the hemibiotrophic fungus Colletotrichum higginsianum.

Thirty-one endophytic bacteria isolated from healthy leaves of Centella asiatica were screened in vitro for their ability to reduce the growth rate and disease incidence of Colletotrichum higginsianum, a causal agent of anthracnose. Isolates of Cohnella sp., Paenibacillus sp. and Pantoea sp. significantly stimulated the growth rate of C. higginsianum MUCL 44942, while isolates of Achromobacter sp., Acinetobacter sp., Microbacterium sp., Klebsiella sp. and Pseudomonas putida had no influence on this plant pathogen. By contrast, Bacillus subtilis BCA31 and Pseudomonas fluorescens BCA08 caused a marked inhibition of C. higginsianum MUCL 44942 growth by 46 and 82 %, respectively. Cell-free culture filtrates of B. subtilis BCA31 and P. fluorescens BCA08 were found to contain antifungal compounds against C. higginsianum MUCL 44942. Inoculation assays on in vitro-cultured plants of C. asiatica showed that foliar application of B. subtilis BCA31, three days before inoculation with C. higginsianum MUCL 44942, significantly reduced incidence and severity of the disease. The role of endophytic bacteria in maintaining the apparent inactivity of C. higginsianum MUCL 44942 in C. asiatica grown in the wild is discussed.

Transcriptional regulation of defence genes and involvement of the WRKY transcription factor in arbuscular mycorrhizal potato root colonization.

The establishment of arbuscular mycorrhizal associations causes major changes in plant roots and affects significantly the host in term of plant nutrition and resistance against biotic and abiotic stresses. As a consequence, major changes in root transcriptome, especially in plant genes related to biotic stresses, are expected. Potato microarray analysis, followed by real-time quantitative PCR, was performed to detect the wide transcriptome changes induced during the pre-, early and late stages of potato root colonization by Glomus sp. MUCL 41833. The microarray analysis revealed 526 up-regulated and 132 down-regulated genes during the pre-stage, 272 up-regulated and 109 down-regulated genes during the early stage and 734 up-regulated and 122 down-regulated genes during the late stage of root colonization. The most important class of regulated genes was associated to plant stress and in particular to the WRKY transcription factors genes during the pre-stage of root colonization. The expression profiling clearly demonstrated a wide transcriptional change during the pre-, early and late stages of root colonization. It further suggested that the WRKY transcription factor genes are involved in the mechanisms controlling the arbuscular mycorrhizal establishment by the regulation of plant defence genes.