Evolutionary history of arbuscular mycorrhizal fungi and genomic signatures of obligate symbiosis.

BACKGROUND: The colonization of land and the diversification of terrestrial plants is intimately linked to the evolutionary history of their symbiotic fungal partners. Extant representatives of these fungal lineages include mutualistic plant symbionts, the arbuscular mycorrhizal (AM) fungi in Glomeromycota and fine root endophytes in Endogonales (Mucoromycota), as well as fungi with saprotrophic, pathogenic and endophytic lifestyles. These fungal groups separate into three monophyletic lineages but their evolutionary relationships remain enigmatic confounding ancestral reconstructions. Their taxonomic ranks are currently fluid. RESULTS: In this study, we recognize these three monophyletic linages as phyla, and use a balanced taxon sampling and broad taxonomic representation for phylogenomic analysis that rejects a hard polytomy and resolves Glomeromycota as sister to a clade composed of Mucoromycota and Mortierellomycota. Low copy numbers of genes associated with plant cell wall degradation could not be assigned to the transition to a plant symbiotic lifestyle but appears to be an ancestral phylogenetic signal. Both plant symbiotic lineages, Glomeromycota and Endogonales, lack numerous thiamine metabolism genes but the lack of fatty acid synthesis genes is specific to AM fungi. Many genes previously thought to be missing specifically in Glomeromycota are either missing in all analyzed phyla, or in some cases, are actually present in some of the analyzed AM fungal lineages, e.g. the high affinity phosphorus transporter Pho89. CONCLUSION: Based on a broad taxon sampling of fungal genomes we present a well-supported phylogeny for AM fungi and their sister lineages. We show that among these lineages, two independent evolutionary transitions to mutualistic plant symbiosis happened in a genomic background profoundly different from that known from the emergence of ectomycorrhizal fungi in Dikarya. These results call for further reevaluation of genomic signatures associated with plant symbiosis.

Rhizophagus irregularis and biochar can synergistically improve the physiological characteristics of saline-alkali resistance of switchgrass.

Inoculation of arbuscular mycorrhizal fungi (AMF) or biochar (BC) application can improve photosynthesis and promote plant growth under saline-alkali stress. However, little is known about the effects of the two combined on growth and physiological characteristics of switchgrass under saline-alkali stress. This study examined the effects of four treatments: (1) no AMF inoculation and no biochar addition (control), (2) biochar (BC) alone, (3) AMF (Rhizophagus irregularis, Ri) alone, and (4) the combination of both (BC+Ri) on the plant biomass, antioxidant enzymes, chlorophyll, and photosynthetic parameters of switchgrass under saline-alkali stress. The results showed that the above-ground, belowground and total biomass of switchgrass in the BC+Ri treatment group was significantly higher (+136.7%, 120.2% and 132.4%, respectively) than in other treatments compared with Control. BC+Ri treatment significantly increased plant leaves' relative chlorophyll content, antioxidant enzyme activity, and photosynthesis parameters. It is worth noting that the transpiration rate, stomatal conductance, net photosynthetic rate, PSII efficiency and other photosynthetic-related indexes of the BC+Ri treatment group were the highest (38% to 54% higher than other treatments). The fitting results of light response and CO2 response curves showed that the light saturation point, light compensation point, maximum carboxylation rate and maximum electron transfer rate of switchgrass in the Ri+BC treatment group were the highest. In conclusion, biochar combined with Ri has potential beneficial effects on promoting switchgrass growth under saline-alkali stress and improving the activity of antioxidant enzymes and photosynthetic characteristics of plants.

Divergent colonization traits, convergent benefits: different species of arbuscular mycorrhizal fungi alleviate Meloidogyne incognita damage in tomato.

Arbuscular mycorrhizal fungi (AMF) can increase plant tolerance and/or resistance to pests such as the root-knot nematode Meloidogyne incognita. However, the ameliorative effects may depend on AMF species. The aim of this work was therefore to evaluate whether four AMF species differentially affect plant performance in response to M. incognita infection. Tomato plants grown in greenhouse conditions were inoculated with four different AMF isolates (Claroideoglomus claroideum, Funneliformis mosseae, Gigaspora margarita, and Rhizophagus intraradices) and infected with 100 second stage juveniles of M. incognita at two different times: simultaneously or 2 weeks after the inoculation with AMF. After 60 days, the number of galls, egg masses, and reproduction factor of the nematodes were assessed along with plant biomass, phosphorus (P), and nitrogen concentrations in roots and shoots and root colonization by AMF. Only the simultaneous nematode inoculation without AMF caused a large reduction in plant shoot biomass, while all AMF species were able to ameliorate this effect and improve plant P uptake. The AMF isolates responded differently to the interaction with nematodes, either increasing the frequency of vesicles (C. claroideum) or reducing the number of arbuscules (F. mosseae and Gi. margarita). AMF inoculation did not decrease galls; however, it reduced the number of egg masses per gall in nematode simultaneous inoculation, except for C. claroideum. This work shows the importance of biotic stress alleviation associated with an improvement in P uptake and mediated by four different AMF species, irrespective of their fungal root colonization levels and specific interactions with the parasite.

Soil compaction reversed the effect of arbuscular mycorrhizal fungi on soil hydraulic properties.

Arbuscular mycorrhizal fungi (AMF) typically provide a wide range of nutritional benefits to their host plants, and their role in plant water uptake, although still controversial, is often cited as one of the hallmarks of this symbiosis. Less attention has been dedicated to other effects relating to water dynamics that the presence of AMF in soils may have. Evidence that AMF can affect soil hydraulic properties is only beginning to emerge. In one of our recent experiments with dwarf tomato plants, we serendipitously found that the arbuscular mycorrhizal fungus (Rhizophagus irregularis 'PH5') can slightly but significantly reduce water holding capacity (WHC) of the substrate (a sand-zeolite-soil mixture). This was further investigated in a subsequent experiment, but there we found exactly the opposite effect as mycorrhizal substrate retained more water than did the non-mycorrhizal substrate. Because the same substrate was used and other conditions were mostly comparable in the two experiments, we explain the contrasting results by different substrate compaction, most likely caused by different pot shapes. It seems that in compacted substrates, AMF may have no effect upon or even decrease the substrates' WHC. On the other hand, the AMF hyphae interweaving the pores of less compacted substrates may increase the capillary movement of water throughout such substrates and cause slightly more water to remain in the pores after the free water has drained. We believe that this phenomenon is worthy of mycorrhizologists' attention and merits further investigation as to the role of AMF in soil hydraulic properties.

Absence of Gigasporales and rarity of spores in a hot desert revealed by a multimethod approach.

Hot deserts impose extreme conditions on plants growing in arid soils. Deserts are expanding due to climate change, thereby increasing the vulnerability of ecosystems and the need to preserve them. Arbuscular mycorrhizal fungi (AMF) improve plant fitness by enhancing plant water/nutrient uptake and stress tolerance. However, few studies have focused on AMF diversity and community composition in deserts, and the soil and land use parameters affecting them. This study aimed to comprehensively describe AMF ecological features in a 5,000 km2 arid hyperalkaline region in AlUla, Saudi Arabia. We used a multimethod approach to analyse over 1,000 soil and 300 plant root samples of various species encompassing agricultural, old agricultural, urban and natural ecosystems. Our method involved metabarcoding using 18S and ITS2 markers, histological techniques for direct AMF colonization observation and soil spore extraction and observation. Our findings revealed a predominance of AMF taxa assigned to Glomeraceae, regardless of the local conditions, and an almost complete absence of Gigasporales taxa. Land use had little effect on the AMF richness, diversity and community composition, while soil texture, pH and substantial unexplained stochastic variance drove these compositions in AlUla soils. Mycorrhization was frequently observed in the studied plant species, even in usually non-mycorrhizal plant taxa (e.g. Amaranthaceae, Urticaceae). Date palms and Citrus trees, representing two major crops in the region, however, displayed a very low mycorrhizal frequency and intensity. AlUla soils had a very low concentration of spores, which were mostly small. This study generated new insight on AMF and specific behavioral features of these fungi in arid environments.

Unraveling the diversity of hyphal explorative traits among Rhizophagus irregularis genotypes.

Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood.

Rhizophagus Irregularis regulates flavonoids metabolism in paper mulberry roots under cadmium stress.

Broussonetia papyrifera is widely found in cadmium (Cd) contaminated areas, with an inherent enhanced flavonoids metabolism and inhibited lignin biosynthesis, colonized by lots of symbiotic fungi, such as arbuscular mycorrhizal fungi (AMF). However, the physiological and molecular mechanisms by which Rhizophagus irregularis, an AM fungus, regulates flavonoids and lignin in B. papyrifera under Cd stress remain unclear. Here, a pot experiment of B. papyrifera inoculated and non-inoculated with R. irregularis under Cd stress was carried out. We determined flavonoids and lignin concentrations in B. papyrifera roots by LC-MS and GC-MS, respectively, and measured the transcriptional levels of flavonoids- or lignin-related genes in B. papyrifera roots, aiming to ascertain the key components of flavonoids or lignin, and key genes regulated by R. irregularis in response to Cd stress. Without R. irregularis, the concentrations of eriodictyol, quercetin and myricetin were significantly increased under Cd stress. The concentrations of eriodictyol and genistein were significantly increased by R. irregularis, while the concentration of rutin was significantly decreased. Total lignin and lignin monomer had no alteration under Cd stress or with R. irregularis inoculation. As for flavonoids- or lignin-related genes, 26 genes were co-regulated by Cd stress and R. irregularis. Among these genes, BpC4H2, BpCHS8 and BpCHI5 were strongly positively associated with eriodictyol, indicating that these three genes participate in eriodictyol biosynthesis and were involved in R. irregularis assisting B. papyrifera to cope with Cd stress. This lays a foundation for further research revealing molecular mechanisms by which R. irregularis regulates flavonoids synthesis to enhance tolerance of B. papyrifera to Cd stress.

Mycorrhizal Symbiosis Can Change the Composition of Secondary Metabolites in Fruits of Solanum nigrum L.

Solanum nigrum is a common weed in arable land, while being used in traditional medicine around the world due to its remarkable levels of valuable secondary metabolites. Agronomic and biological techniques can alter the production of a specific metabolite by influencing plant growth and metabolism. The effects of colonization with three arbuscular mycorrhizal fungi (AMF), including Funneliformis mosseae, Rhizoglomus intraradices, and Rhizoglomus fasciculatum, on the chemical composition of S. nigrum fruits were evaluated by gas chromatography-mass spectrometry (GC-MS) analysis. More than 100 different chemical constituents were evaluated by GC-MS. Our study revealed that the levels of phenols (quinic acid), benzenes (hydroquinone), sulfur-containing compounds, lactone and carboxylic acids were improved by R. intraradices. In contrast, hydroxymethylfurfural increased by 68 % in R. fasciculatum inoculated with uninoculated S. nigrum plants, and this species was also the most efficient in inducing sugar compounds (D-galactose, lactose, and melezitose). Our results suggest that AMF colonization is an effective biological strategy that can alter the chemical composition and improve the medicinal properties of S. nigrum.

Glomus versiforme and intercropping with Sphagneticola calendulacea decrease Cd accumulation in maize.

Little information is available on the influence of the compound use of intercropping (IN) and arbuscular mycorrhizal fungus (AMF) on Cd accumulation and the expression of Cd transporter genes in two intercropped plants. A pot experiment was conducted to study the influences of IN and AMF-Glomus versiforme on growth and Cd uptake of two intercropped plants-maize and Cd hyperaccumulator Sphagneticola calendulacea, and the expression of Cd transporter genes in maize in Cd-polluted soils. IN, AMF and combined treatments of IN and AMF (IN + AMF) obviously improved biomass, photosynthesis and total antioxidant capacities of two plants. Moreover, single and compound treatments of IN and AMF evidently reduced Cd contents in maize, and the greatest decreases appeared in the compound treatment. However, Cd contents of S. calendulacea in IN, AMF and IN + AMF groups were notably improved. Furthermore, the single and compound treatments of IN and AMF significantly downregulated the expression levels of Nramp1, HMA1, ABCC1 and ABCC10 in roots and leaves, and the largest decreases were observed in the combined treatment. Our work first revealed that the combined use of IN and AMF appeared to have a synergistic effect on decreasing Cd content by downregulating the expression of Cd transporter genes in maize.

Transcriptome analysis shows that Glomus versiforme decrease the accumulation and toxicity of cadmium in Ipomoea aquatic Forsk.

So far, the physiological and molecular mechanisms of the impact of arbuscular mycorrhizal fungus (AMF) on Cd absorption, transport and detoxification in Ipomoea aquatica (water spinach) are still unclear. In the present study, a pot experiment was performed to investigate the impact of AMF-Glomus versiforme (Gv) on the photosynthetic characteristics, Cd uptake, antioxidative system and transcriptome in water spinach in the soils supplemented with 5 mg Cd kg-1. Gv inoculation improved significantly the photosynthetic characteristics and growth of water spinach. Furthermore, Gv colonization significantly promoted the activities of catalase (CAT), peroxidase (POD) and glutathione reductase (GR), contents of glutathione (GSH) and ascorbic acid (AsA), and the total antioxidant capacity (TCA), but decreased malondialdehyde (MDA) content in water spinach. In addition, Gv inoculation significantly increased pH in rhizosphere soils and decreased the Cd concentrations and uptakes in water spinach. Importantly, 2670 differentially expressed genes (DEGs) were screened in water spinach root colonized with Gv in 5 mg Cd kg-1 soil, of which 2008 DEGs were upregulated and 662 DEGs were downregulated. Especially, the expression levels of POD, CAT, GR, dehydroascorbate reductase 2 (DHAR2), glutathione S-transferase U8 (GSTU8) and glutathione synthetase (GSHS) and cytochrome P450 (Cyt P450) genes were significantly up-regulated in water spinach inoculated with Gv. Meanwhile, the plant cadmium resistance protein 2 (PCR2), metal tolerance protein 4 (MTP4), ATP-binding cassette transporter C family member (ABCC), ABC-yeast cadmium factor 1 (ABC-YCF1) and metallothionein (MT) genes were also up-regulated in mycorrhizal water spinach. Our results firstly elucidated the mechanism by which AMF reduced the uptake and phytotoxicity of Cd in water spinach through a transcriptome analysis.

Seven-year long-term inoculation with Funneliformis mosseae increases maize yield and soil carbon storage evidenced by in situ 13C-labeling in a dryland.

Arbuscular mycorrhizal fungi (AMF) establish symbiotic relationships with roots of most plants, contributing to plant water uptake and soil carbon (C) sequestration. However, the interactive contribution and of long-term field AMF inoculation and water conservation on maize yield and soil organic carbon (SOC) sequestration in drylands remain largely unknown. After 7-year long-term field inoculation with AMF Funneliformis mosseae, AMF suppression by fungicide benomyl, and no-AMF/no-benomyl control, and two water conservation practices of half-film and full-film mulching (∼50 % and ∼100 crop planted area covered with plastic film), this study thus applied in situ 13CO2-C labeling and high-throughput sequencing to quantify newly photosynthetically assimilated C into different soil C pools including soil aggregates and respiration, and their effects on maize growth and productivity. Results showed that 7-year long-term AMF inoculation significantly increased the relative abundance of F. mosseae in rhizosphere soil and root AMF colonization, indicating that F. mosseae successfully dominated in AMF communities. Compared to no-AMF/no-benomyl control, AMF colonization significantly increased shoot biomass and maize yield by 17.9 % and 20.3 % while mitigated the less water conservation effects of half-film mulching on maize performance. The SOC content under field AMF inoculation SOC was increased from 7.9 to 8.4 g kg-1 and also the mean weight diameter of aggregates (1.21 to 1.35), e.g. aggregate stability. After 1 and/or 40 days 13C labeling, the enhanced 13C translocations into macro-aggregates with decreased 13C emissions from microbial decomposition under field AMF inoculation had contributed to SOC conservation in bulk soil. These results suggest that AMF inoculation in dryland crops is promising to increase crop yield while promoting more atmospheric CO2 fixation in soil aggregates. A long-term field AMF inoculation will enhance our understanding of applying beneficial mycorrhizal fungi to enhance soil C sequestration and also crop yield via plant-fixed atmospheric CO2 in semi-arid and arid farmlands.

Functionality of arbuscular mycorrhizal fungi varies across different growth stages of maize under drought conditions.

Physio-biochemical regulations governing crop growth period are pivotal for drought adaptation. Yet, the extent to which functionality of arbuscular mycorrhizal fungi (AM fungi) varies across different stages of maize growth under drought conditions remains uncertain. Therefore, periodic functionality of two different AM fungi i.e., Rhizophagus irregularis SUN16 and Glomus monosporum WUM11 were assessed at jointing, silking, and pre-harvest stages of maize subjected to different soil moisture gradients i.e., well-watered (80% SMC (soil moisture contents)), moderate drought (60% SMC), and severe drought (40% SMC). The study found that AM fungi significantly (p < 0.05) affected various morpho-physiological and biochemical parameters at different growth stages of maize under drought. As the plants matured, AM fungi enhanced root colonization, glomalin contents, and microbial biomass, leading to increased nutrient uptake and antioxidant activity. This boosted AM fungal activity ultimately improved photosynthetic efficiency, evident in increased photosynthetic pigments and photosynthesis. Notably, R. irregularis and G. monosporum improved water use efficiency and mycorrhizal dependency at critical growth stages like silking and pre-harvest, indicating their potential for drought resilience to stabilize yield. The principal component analysis highlighted distinct plant responses to drought across growth stages and AM fungi, emphasizing the importance of early-stage sensitivity. These findings underscore the potential of incorporating AM fungi into agricultural management practices to enhance physiological and biochemical responses, ultimately improving drought tolerance and yield in dryland maize cultivation.

Alternative splicing regulation in plants by SP7-like effectors from symbiotic arbuscular mycorrhizal fungi.

Most plants in natural ecosystems associate with arbuscular mycorrhizal (AM) fungi to survive soil nutrient limitations. To engage in symbiosis, AM fungi secrete effector molecules that, similar to pathogenic effectors, reprogram plant cells. Here we show that the Glomeromycotina-specific SP7 effector family impacts on the alternative splicing program of their hosts. SP7-like effectors localize at nuclear condensates and interact with the plant mRNA processing machinery, most prominently with the splicing factor SR45 and the core splicing proteins U1-70K and U2AF35. Ectopic expression of these effectors in the crop plant potato and in Arabidopsis induced developmental changes that paralleled to the alternative splicing modulation of a specific subset of genes. We propose that SP7-like proteins act as negative regulators of SR45 to modulate the fate of specific mRNAs in arbuscule-containing cells. Unraveling the communication mechanisms between symbiotic fungi and their host plants will help to identify targets to improve plant nutrition.

Inoculation with Funneliformis mosseae promotes selenium accumulation and transformation in pepper (Capsicum annuum L.).

Selenium (Se) is one of the fifteen essential nutrients required by the human body. Mycorrhizal microorganisms play a crucial role in enhancing selenium availability in plants. However, limited research exists on the impact of arbuscular mycorrhizal fungi (AMF) on selenium accumulation and transport in pepper plants. This study employed a pot experiment to investigate the changes in pepper plant growth, selenium accumulation, and transformation following inoculation with AMF and varying concentrations of exogenous selenium. The results indicate that exogenous selenium application in pepper has dual effects. At low concentrations (≤8 mg L⁻1), it promotes growth and nutrient accumulation, whereas high concentrations (>16 mg L⁻1) inhibit these processes. AMF inoculation positively influences selenium accumulation and transport in peppers, significantly increasing yield per plant by 17.89%, vitamin C content by 67.36%, flavonoid content by 43.26%, capsaicin content by 14.82%, DPPH radical scavenging rate by 18.18%, and ABTS radical scavenging rate by 27.81%. Additionally, it significantly reduces selenocysteine methyltransferase (SMT) enzyme activity, while minimally affecting ATP sulfurylase (ATPS) and adenosyl sulfate reductase (APR) enzyme activities. The combined treatment of AMF and 8 mg L⁻1 exogenous selenium has been proven to be the most effective for selenium enrichment in peppers, offering new insights into utilizing exogenous selenium and AMF inoculation to enhance selenium content in peppers.

Growth-promoting bacteria and arbuscular mycorrhizal fungus enhance maize tolerance to saline stress.

Climate change intensifies soil salinization and jeopardizes the development of crops worldwide. The accumulation of salts in plant tissue activates the defense system and triggers ethylene production thus restricting cell division. We hypothesize that the inoculation of plant growth-promoting bacteria (PGPB) producing ACC (1-aminocyclopropane-1-carboxylate) deaminase favors the development of arbuscular mycorrhizal fungi (AMF), promoting the growth of maize plants under saline stress. We investigated the efficacy of individual inoculation of PGPB, which produce ACC deaminase, as well as the co-inoculation of PGPB with Rhizophagus clarus on maize plant growth subjected to saline stress. The isolates were acquired from the bulk and rhizospheric soil of Mimosa bimucronata (DC.) Kuntze in a temporary pond located in Pernambuco State, Brazil. In the first greenhouse experiment, 10 halophilic PGPB were inoculated into maize at 0, 40 and 80 mM of NaCl, and in the second experiment, the PGPB that showed the best performance were co-inoculated with R. clarus in maize under the same conditions as in the first experiment. Individual PGPB inoculation benefited the number of leaves, stem diameter, root and shoot dry mass, and the photosynthetic pigments. Inoculation with PGPB 28-10 Pseudarthrobacter enclensis, 24-1 P. enclensis and 52 P. chlorophenolicus increased the chlorophyll a content by 138%, 171%, and 324% at 0, 40 and 80 mM NaCl, respectively, comparing to the non-inoculated control. We also highlight that the inoculation of PGPB 28-10, 28-7 Arthrobacter sp. and 52 increased the content of chlorophyll b by 72%, 98%, and 280% and carotenoids by 82%, 98%, and 290% at 0, 40 and 80 mM of NaCl, respectively. Co-inoculation with PGPB 28-7, 46-1 Leclercia tamurae, 70 Artrobacter sp., and 79-1 Micrococcus endophyticus significantly increased the rate of mycorrhizal colonization by roughly 50%. Furthermore, co-inoculation promoted a decrease in the accumulation of Na and K extracted from plant tissue, with an increase in salt concentration, from 40 mM to 80 mM, also favoring the establishment and development of R. clarus. In addition, co-inoculation of these PGPB with R. clarus promoted maize growth and increased plant biomass through osmoregulation and protection of the photosynthetic apparatus. The tripartite symbiosis (plant-fungus-bacterium) is likely to reprogram metabolic pathways that improve maize growth and crop yield, suggesting that the AMF-PGPB consortium can minimize damages caused by saline stress.

Investigating the simultaneous effect of chitosan and arbuscular mycorrhizal fungi on growth, phenolic compounds, PAL enzyme activity and lipid peroxidation in Salvia nemorosa L.

Considering the importance of Salvia nemorosa L. in the pharmaceutical and food industries, and also beneficial approaches of arbuscular mycorrhizal fungi (AMF) symbiosis and the use of bioelicitors such as chitosan to improve secondary metabolites, the aim of this study was to evaluate the performance of chitosan on the symbiosis of AMF and the effect of both on the biochemical and phytochemical performance of this plant and finally introduced the best treatment. Two factors were considered for the factorial experiment: AMF with four levels (non-inoculated plants, Funneliformis mosseae, Rhizophagus intraradices and the combination of both), and chitosan with six levels (0, 50, 100, 200, 400 mg L-1 and 1% acetic acid). Four months after treatments, the aerial part and root length, the levels of lipid peroxidation, H2O2, phenylalanine ammonia lyase (PAL) activity, total phenol and flavonoid contents and the main secondary metabolites (rosmarinic acid and quercetin) in the leaves and roots were determined. The flowering stage was observed in R. intraradices treatments and the highest percentage of colonization (78.87%) was observed in the treatment of F. mosseae × 400 mg L-1 chitosan. Furthermore, simultaneous application of chitosan and AMF were more effective than their separate application to induce phenolic compounds accumulation, PAL activity and reduce oxidative compounds. The cluster and principal component analysis based on the measured variables indicated that the treatments could be classified into three clusters. It seems that different treatments in different tissues have different effects. However, in an overview, it can be concluded that 400 mg L-1 chitosan and F. mosseae × R. intraradices showed better results in single and simultaneous applications. The results of this research can be considered in the optimization of this medicinal plant under normal conditions and experiments related to abiotic stresses in the future.

Mycorrhizal association in gametophytes and sporophytes of the fern Pteris vittata (Pteridaceae) with Glomus intraradices

Ferns, which are usually colonizing different environments and their roots frequently present mycorrhization, have two adult stages in their life cycle, the sporophytic and the gametophytic phase. This paper describes the experimental mycorrhizal association between Pteris vittata leptosporangiate fern and a strain of Glomus intraradices during the life cycle of the fern, from spore germination to the development of a mature sporophyte. The aim of this study was to compare the colonization pattern of in vitro cultures of G. intraradices along the fern life cycle with those found in nature. For this, mature spores were obtained from fertile P. vittata fronds growing in walls of Buenos Aires city, Argentina. Roots were stained and observed under the light microscope for arbuscular mycorrhizal colonization. Approximately, 75 fern spores were cultured in each pot filled with a sterile substrate and G. intraradices (BAFC N° 51.331) as inoculum on the surface. After germination took place, samples were taken every 15 days until the fern cycle was completed. In order to determine colonization dynamics each sample was observed under optical and confocal microscope after staining. Gametophyte was classified as Adiantum type. Male and female gametangia were limited to the lower face, mycorrhizal colonization started when they were differentiated and took place through the rhizoids. Spores and vesicles were not found in this cycle stage. Paris-type mycorrhizal colonization was established in the midrib and in the embrionary foot. It was colonized by external mycelium. When the first root was developed soil inoculum colonized de novo this structure and Arum-type colonization was observed. This study proves that the type of colonization is determined by the structure of the host, not by the fungus. Both the gametophyte and embryo foot have determined growth and Paris-type colonization, while, sporophyte roots have undetermined growth and Arum-type colonization. The structures found in vitro cultures were highly similar to those found under natural conditions. Rev. Biol. Trop. 60 (2): 857-865. Epub 2012 June 01.

Los helechos presentan dos etapas en su ciclo de vida, una fase esporofítica y una gametofítica. Estos por lo general pueden colonizar diferentes ambientes y frecuentemente presentan raíces micorrizadas. Este estudio describe la asociación experimental entre Pteris vittata, un helecho leptosporangiado y una cepa de Glomus intraradices durante el ciclo de vida del helecho, desde la germinación de las esporas hasta el desarrollo del esporofito maduro. El objetivo de este estudio fue comparar los patrones de colonización de G. intraradices a lo largo de todo el ciclo de vida del helecho con los tipos encontrados en la naturaleza. Las esporas maduras fueron obtenidas de frondes fértiles de P. vittata que crecen sobre las paredes de la ciudad de Buenos Aires, Argentina. Las raíces se tiñeron y fueron observadas bajo microscopio óptico para el estudio de la colonización micorrízica. Aproximadamente 75 esporas de helecho se cultivaron en macetas con un sustrato estéril y con un inóculo de G. intraradices (N° 51.331 BAFC) en la superficie. Después de la germinación, se tomaron muestras cada 15 días hasta que se completó el ciclo de vida del helecho. Con el fin de determinar la dinámica de la colonización, cada muestra se observó con el microscopio óptico y el microscopio de confocal luego de la tinción correspondiente. El gametofito fue clasificado como del tipo “Adiantum”. Los gametangios femeninos y masculinos se desarrollaron en la cara inferior del mismo. La micorrización comenzó cuando los gametangios estaban ya diferenciados y la colonización se produjo a través de los rizoides. Las esporas y las vesículas no se encontraron en esta fase del ciclo. La micorrizacion tipo Paris se observó sobre la línea de la nervadura central. El pie del esporofito fue colonizado por el micelio externo. Cuando la raíz se desarrolló, se colonizó “de novo”, y se observó una colonización de tipo Arum. Este estudio demuestra que el tipo de colonización está determinado por la estructura del helecho y no por el hongo. Tanto el gametofito como el pie del embrión tienen crecimiento definido y colonización tipo Paris, mientras que las raíces del esporofito presentan un crecimiento indeterminado y una colonización tipo Arum. Las estructuras que se encontraron bajo cultivo coinciden con las que se encontraron en condiciones naturales.

Mycorrhizal-like interaction between gametophytes and young sporophytes of the fern Dryopteris muenchii (Filicales) and its fungal endophyte

The morphology of a Glomus-like fungus-host interaction in chlorophyllous gametophytes and young apogamic sporophytes of Dryopteris muenchii A.R. Sm. was studied from ferns cultivated in laboratory, using soil as substrate. An aseptate fungus colonized the gametophytes’ tissue through the rhizoids, developing vesicles. The fungus penetrated the young sporophytes primary roots by developing appressoria. It spread forming inter- and intra-cellular hyphae through the epidermis and the outermost cortical cell layers, where it formed vesicles, hyphal coils-like and arbuscules. The fungus hyphae never colonized the gametophyte-sporophyte cellular junction. The fungal structures observed on D. muenchii during this study, are rather similar to those reported for the plant host-arbuscular mycorrhizal fungus (AMF) interaction, where the AMF described belonged to Phylum Glomeromycota. Therefore, this study is a contribution to the scarce knowledgement of the association between AMF and chlorophyllous gametophytes and young apogamic sporophytes of ferns. Rev. Biol. Trop. 56 (3): 1101-1107. Epub 2008 September 30.

Se describe la morfología de un hongo endófito afín al género Glomus, como colonizador de gametofitos clorofílicos y de esporofitos apogámicos jóvenes del helecho Dryopteris muenchii A.R. Sm.; el estudio se llevó a cabo con helechos cultivados en el laboratorio y utilizando tierra como substrato. El tejido del gametofito fue colonizado, a través de los rizoides, por un hongo miceliar aseptado, el cual formó vesículas. El hongo logró penetrar las raíces primarias de los esporofitos jóvenes desarrollando apresorios. El hongo se dispersó formando hifas inter- e intra-celulares a través de la epidermis y de la capa de células corticales más externas, donde produjo vesículas, estructuras similares a ovillos y arbúsculos. Las hifas del hongo nunca colonizaron las células de la unión entre el gametofito y el esporofito. Las estructuras observadas durante este estudio en D. muenchii, son muy similares a las de la interacción planta hospedera-hongo micorrícico arbuscular (HMA), en donde el HMA descrito corresponde al Phylum Glomeromycota. Por lo anterior, este estudio es una contribución al escaso conocimiento que se tiene sobre la asociación entre los HMA y gametofitos clorofílicos y esporofitos apogámicos de helechos jóvenes.