Different behavior of two strains of the arbuscular mycorrhizal fungus Rhizophagus intraradices on Senecio bonariensis Hook. & Arn. against heavy metal soil pollution: a pilot-scale test.

Arbuscular mycorrhizal fungi (AMF) have different biological mechanisms to alleviate stressful conditions in heavy metals (HMs) polluted soil. These mechanisms were widely assessed under controlled/greenhouse conditions, but scarcely studied at pilot or territory scale. The aim of this study was to evaluate the response of two Rhizophagus intraradices strains isolated from soils with different histories of pollution, in association with Senecio bonariensis plants, growing in an engineering vegetal depuration module filled with artificially HMs polluted substrate. Plants inoculated with GC3 strain uptook low amounts of HMs and translocated them to shoot biomass. Heavy metals (Mg, Zn, Mn, Cr, Cu and Ni) and macronutrients (Ca, K, S and P) were accumulated in roots of S. bonariensis when inoculated with GB8 strain, limiting their translocation to the shoot. Uninoculated plants showed high translocation of all studied elements to shoot tissues. Concluding, tested R. intraradices strains have exhibited different phytoprotection mechanisms under extremely toxic concentrations of HMs. Moreover, the development of the assay at such a high Technological Readiness Level represents a novel contribution in this field of study.

Mycorrhizal stress alleviation in Senecio bonariensis Hook & Arn growing in urban polluted soils.

Loss of biodiversity and accumulation of contaminants in urban soils and water bodies cause serious issues in metropolitan areas. The Matanza-Riachuelo river basin (metropolitan area of Buenos Aires, Argentina) is one of the most environmentally degraded regions in the world. Senecio bonariensis Hook & Arn (Asteraceae) grows in the periodically flooded soils of this wetland. This plant concentrates potentially toxic trace elements (PTEs) in its tissues and establishes symbiosis with arbuscular mycorrhizal (AM) fungi that collaborate with PTE phytostabilization in soils. The objective of this work was to evaluate tolerance and stress alleviation of AM-colonized S. bonariensis when transplanting and exposing to highly polluted environmental conditions of the river basin. Plants were initially inoculated with different AM strains and maintained in greenhouse conditions. After 6 mo, they were transplanted to the field. These plants showed a more equal distribution between shoot and root biomass production in comparison to field spontaneous S. bonaerensis plants. Plants in earlier contact with native soil inoculum showed positive correlation with phosphorus content and a significant increase of vesicle frequency. Plants belatedly contacted with native inoculum in the field (control) showed a higher catalase level that was positively correlated with the total colonization frequency and chlorophyll content. The ability to establish symbiosis with Rhizophagus intraradices (strain GC3), commonly used in the formulation of biofertilizers, was also analyzed. Plants inoculated with GC3 at the beginning of the assay showed lower colonization and were less efficient in the field. The preservation of spontaneous native plants with ornamental value and bioaugmentation of their associated microbiome can contribute to the stabilization of contaminants in soils.

Arbuscular mycorrhizal fungi in heavy metal highly polluted soil in the Riachuelo river basin Arbuscular mycorrhizal fungi in heavy metal highly polluted soil / Hongos formadores de micorrizas arbusculares en el suelo extremadamente contaminado con metales pesados de la cuenca del río Riachuelo

Abstract The Riachuelo river basin (RRB) is considered one of the most polluted environments in the world. Knowledge of arbuscular mycorrhizal fungi (AMF) adapted to this extremely polluted environment is important for the establishment of future soil restoration projects. This work aims to make a first list of AMF species present on the RRB. Soil and root samples were randomly taken in an area of approximately 1500 m2, mycorrhization percentages were evaluated. AMF species were detected by molecular and morphological techniques. Sixteen AMF morphological species and 64 molecular species were reported in this work. Dominikia iranica, Funneliformis constrictum, Funneliformis mosseae, Rhizophagus intraradices, Rhizophagus irregularis and Septoglomus viscosum were detected by both techniques while Claroideoglomus sp. was only detected by pyrosequencing. The list of species reported in this work represents the first description of the RRB AMF community.

Resumen La cuenca del río Riachuelo (CRR) es considerada uno de los ambientes más contaminados del mundo. Conocer los hongos formadores de micorrizas arbusculares (HFMA) adaptados a este ambiente extremadamente contaminado es importante para el establecimiento de futuros proyectos de restauración de suelos. Este trabajo se propuso hacer una primera lista de especies de HFMA presentes en la CRR. Se tomaron muestras de suelo y raíces al azar en un área de aproximadamente 1500 m2 y se evaluaron los porcentajes de micorrización. La identificación de especies de HFMA se basó en técnicas moleculares y morfológicas. Se detectaron 16 especies morfológicas y 64 especies moleculares de HFMA. Dominikia iranica, Funneliformis constrictum, Funneliformis mosseae, Rhizophagus intraradices, Rhizophagus irregularis y Septoglomus viscosum se detectaron mediante ambas técnicas, mientras que Claroideoglomus sp. solo fue detectado por pirosecuenciación. La lista de especies reportada en este trabajo representa la primera descripción de la comunidad de HFMA de la CRR.

Arbuscular mycorrhizal fungi in heavy metal highly polluted soil in the Riachuelo river basin.

The Riachuelo river basin (RRB) is considered one of the most polluted environments in the world. Knowledge of arbuscular mycorrhizal fungi (AMF) adapted to this extremely polluted environment is important for the establishment of future soil restoration projects. This work aims to make a first list of AMF species present on the RRB. Soil and root samples were randomly taken in an area of approximately 1500m2, mycorrhization percentages were evaluated. AMF species were detected by molecular and morphological techniques. Sixteen AMF morphological species and 64 molecular species were reported in this work. Dominikia iranica, Funneliformis constrictum, Funneliformis mosseae, Rhizophagus intraradices, Rhizophagus irregularis and Septoglomus viscosum were detected by both techniques while Claroideoglomus sp. was only detected by pyrosequencing. The list of species reported in this work represents the first description of the RRB AMF community.

Arbuscular Mycorrhizal Fungal Association in Genetically Modified Drought-Tolerant Corn.

The commercial use of genetically modified (GM) plants has significantly increased worldwide. The interactions between GM plants and arbuscular mycorrhizal (AM) fungi are of considerable importance given the agricultural and ecological role of AM and the lack of knowledge regarding potential effects of drought-tolerant GM corn ( L.) on AM fungal symbiosis. This work studied AM fungal colonization in five corn lines growing under two different irrigation regimes (30 and 100% of soil field capacity [SFC]). Four of the lines were GM corn, and two of these were drought tolerant. The experiment was conducted for 60 d in a growth chamber under constant irrigation, after which mycorrhization, corn biomass, and days to plant senescence (DTS) were evaluated. Arbuscular mycorrhizal fungal species of the order were predominant in the soil inocula. At the end of the experiment, all plants showed AM colonization. Mycorrhization was higher at 30% SFC than at 100% SFC. Within the same corn line, the AM fungi produced more vesicles in plant roots under drought stress. Among treatments, DTS varied significantly, and drought-tolerant GM corn lines survived longer than the wild-type corn when maintained at 100% SFC. Corn biomass did not vary among treatments, and no correlations were found between DTS or biomass and mycorrhization. We conclude that overexpression of the gene in corn plants under the experimental conditions of this study did not affect AM fungal infectivity and improved the tolerance of the corn to drought stress.

Mycorrhizal fungi isolated from native terrestrial orchids of pristine regions in Cordoba (Argentina).

Orchidaceae is a highly dependent group on the Rhizoctonia complex that includes Ceratorhiza, Moniliopsis, Epulorhiza and Rhizoctonia, for seed germination and the development of new orchid plants. Thus, the isolation and identification of orchid mycorrhizal fungi are important to understand the orchid-fungus relationship, which can lead to the development of efficient conservation strategies by in vivo germination of seeds from endangered orchid plants. The aim of our work was to isolate and characterize the different mycorrhizal fungi found in roots of terrestrial orchids from Cordoba (Argentina), and, to learn about the natural habit and fungal associations in the Chaco Serrano woodland pristine region. In this study, bloomed orchid root and rhizosphere soil samples were obtained in two times from Valle de Punilla during spring of 2007; samples were kept in plastic bags until processed within 48 hours, and mycorrhizal condition confirmed assessing peloton presence. A total of 23 isolates of the orchideous mycorrhizal Rhizoctonia complex were obtained. The isolates were studied based on morphological characters and ITS-rDNA sequences. Morphological characteristics as color of colonies, texture, growth rate, hyphal diameter and length and presence of sclerotia were observed on culture media. To define the number of nuclei per cell, the isolates were grown in Petri dishes containing water-agar (WA) for three days at 25 degrees C and stained with Safranine-O solution. The mycorrhizal fungi were grouped into binucleate (MSGib, 10 isolates) and multinucleate (MSGim, 13 isolates) based on morphological characteristics of the colonies. We obtained the ITS1-5.8s-ITS4 region that was amplified using primers ITSI and ITS4. Based on DNA sequencing, isolates Q23 and Q29 were found to be related to species of Ceratobasidium. Isolates Q24 and Q4 were related to the binucleated anastomosis group AG-C of Rhizoctonia sp. The rest of the isolates grouped in the Ceratobasidium clade without grouping. From our knowledge this is the first report of the asso- ciation of the AG-C testers with terrestrial orchids. A high specificity was observed in the symbiotic relationship. As the mycorrhizal fungal isolates were obtained from native orchids, they could be incorporated in conservation programes of endangered orchids in Argentina.

Mycorrhizal fungi isolated from native terrestrial orchids of pristine regions in Córdoba (Argentina) / Hongos micorrízicos aislados de orquídeas terrestres nativas de regiones pristinas en Córdoba (Argentina)

Orchidaceae is a highly dependent group on the Rhizoctonia complex that includes Ceratorhiza, Moniliopsis, Epulorhiza and Rhizoctonia, for seed germination and the development of new orchid plants. Thus, the isolation and identification of orchid mycorrhizal fungi are important to understand the orchid-fungus relationship, which can lead to the development of efficient conservation strategies by in vivo germination of seeds from endangered orchid plants. The aim of our work was to isolate and characterize the different mycorrhizal fungi found in roots of terrestrial orchids from Córdoba (Argentina), and, to learn about the natural habit and fungal associations in the Chaco Serrano woodland pristine region. In this study, bloomed orchid root and rhizosphere soil samples were obtained in two times from Valle de Punilla during spring of 2007; samples were kept in plastic bags until processed within 48 hours, and mycorrhizal condition confirmed assessing peloton presence. A total of 23 isolates of the orchideous mycorrhizal Rhizoctonia complex were obtained. The isolates were studied based on morphological characters and ITS-rDNA sequences. Morphological characteristics as color of colonies, texture, growth rate, hyphal diameter and length and presence of sclerotia were observed on culture media. To define the number of nuclei per cell, the isolates were grown in Petri dishes containing water-agar (WA) for three days at 25°C and stained with Safranine-O solution. The mycorrhizal fungi were grouped into binucleate (MSGib, 10 isolates) and multinucleate (MSGim, 13 isolates) based on morphological characteristics of the colonies. We obtained the ITS1-5.8s-ITS4 region that was amplified using primers ITS1 and ITS4. Based on DNA sequencing, isolates Q23 and Q29 were found to be related to species of Ceratobasidium. Isolates Q24 and Q4 were related to the binucleated anastomosis group AG-C of Rhizoctonia sp. The rest of the isolates grouped in the Ceratobasidium clade without grouping. From our knowledge this is the first report of the association of the AG-C testers with terrestrial orchids. A high specificity was observed in the symbiotic relationship. As the mycorrhizal fungal isolates were obtained from native orchids, they could be incorporated in conservation programes of endangered orchids in Argentina.

La Familia Orchidaceae se encuentra estrechamente relacionada con hongos micorrízicos que pertenecen al complejo Rhizoctonia, e incluyen los géneros Ceratorhiza, Moniliopsis, Epulorhiza y Rhizoctonia. Esta asociación es esencial para el desarrollo de nuevas plantas ya que favorecen el proceso de germinación de las semillas. Por lo tanto, el conocimiento de la naturaleza de esta interacción es importante para que los resultados de los programas de conservación de orquídeas sean efectivos. La fragmentación del bosque Chaqueño Serrano en el centro de Argentina, ha alcanzado un punto crítico en los últimos años, afectando el funcionamiento del ecosistema. El objetivo de este trabajo fue: a) aislar y caracterizar hongos micorrízicos presentes en orquídeas terrestres de la provincia de Córdoba (Argentina) y b) conocer el hábitat natural y las asociaciones fúngicas que se establecen en esta región prístina. A partir de las raíces de orquídeas terrestres, se obtuvieron 23 aislamientos de hongos micorrízicos que pertenecen al complejo Rhizoctonia. Estos aislamientos fueron caracterizados con base en caracteres morfológicos y moleculares. Las características morfológicas (color y textura de las colonias, cinética de crecimiento, diámetro y largo de la hifa y presencia de esclerocios) fueron observados en PDA y MEA a 25ºC. El número de núcleos por célula se observó en cultivos crecidos en AA (agar-agua) y teñidos con una solución de Safranine-O. La región ITS se amplificó usando los primers ITS1 e ITS4. Con base en las características morfológicas de la colonia, los aislamientos fueron agrupados en binucleados (MSGib) y multinucleados (MSGim). De acuerdo al cladograma obtenido con las secuencias de ADN, los aislamientos Q23 y Q29 están relacionados a especies de Ceratobasidium, aisladas de raíces de orquídeas. Los aislamientos Q24 y Q4 se asocian con el grupo de anastomosis de Rhizoctonia AG-C. Finalmente, se observó una alta variabilidad en el grado de especificidad existente en la simbiosis que se establece entre las raíces de estas orquídeas terrestres y los aislamientos obtenidos a partir de ellas. Este es el primer reporte de la asociación entre el grupo de anastomosis AG-C y orquídeas terrestres. Dado que estos aislamientos se obtuvieron de orquídeas terrestres nativas, podrían ser incorporados como nuevos patrones para micorrizas de orquídeas terrestres en Argentina. Este trabajo contribuye al conocimiento de la relación simbiótica que se establece entre orquídeas y hongos micorrízicos, así como también al desarrollo de estrategias de conservación de orquídeas terrestres nativas del bosque Chaco Serrano.

Growth dynamics of geographically different arbuscular mycorrhizal fungal isolates belonging to the 'Rhizophagus clade' under monoxenic conditions.

The growth dynamics of extraradical mycelium and spore formation of 14 "Rhizophagus" isolates from different sites in Argentina were evaluated under monoxenic conditions. A modified Gompertz model was used to characterize the development of mycelium and spores for each isolate under the same conditions. The lag time, maximal growth rate and total quantity of both extraradical hyphae and spores were determined. Wide variability among isolates was detected, and all growth parameters were significantly altered by fungal isolate. Discriminant analysis differentiated isolates primarily based on the extent of extraradical hyphae produced, yet such differences did not conclusively correspond to phylogenetic relationships among closely related isolates based on partial SSU sequences. Given that the "Rhizophagus" isolates were grown under controlled conditions for many generations, the expression of phenotypic variability could be attributed to genetic differences that are not completely resolved by phylogenetic analysis employing the small ribosomal gene.

Evaluation of arbuscular mycorrhizal fungi capacity to alleviate abiotic stress of olive (Olea europaea L.) plants at different transplant conditions.

The capacity of roots to sense soil physicochemical parameters plays an essential role in maintaining plant nutritional and developmental functions under abiotic stress. These conditions generate reactive oxygen species (ROS) in plant tissues causing oxidation of proteins and lipids among others. Some plants have developed adaptive mechanisms to counteract such adverse conditions such as symbiotic association with arbuscular mycorrhizal fungi (AMF). AMF enhance plant growth and improve transplant survival by protecting host plants against environmental stresses. The aim of this study was to evaluate the alleviation of transplanting stress by two strains of Rhizophagus irregularis (GC2 and GA5) in olive. Our results show that olive plants have an additional energetic expense in growth due to an adaptative response to the growing stage and to the mycorrhizal colonization at the first transplant. However, at the second transplant the coinoculation improves olive plant growth and protects against oxidative stress followed by the GA5-inoculation. In conclusion, a combination of two AMF strains at the beginning of olive propagation produces vigorous plants successfully protected in field cultivation even with an additional cost at the beginning of growth.

Differential effects of two strains of Rhizophagus intraradices on dry biomass and essential oil yield and composition in Calamintha nepeta.

The aim of this work was to determine the effects of two geographically different strains of Rhizophagus intraradices (M3 and GA5) on the total biomass and essential oil (EO) yield and composition of Calamintha nepeta, with or without phosphorus (P) fertilization, under greenhouse conditions. The plant biomass was not significantly affected by any of the treatments, showing higher values in control plants. Strains had a differential response in their root colonization rates: M3 reduced these parameters while GA5 did not modify them. Both strains affected EO yield in absence of P fertilization: M3 promoted EO yield in C. nepeta plants and GA5 resulted in negative effects. The percentage composition of EO was not significantly modified by either strain or P fertilization. M3 strain could be a potential fungal bioinoculant for production and commercialization of C. nepeta in the aromatic plant market.

Differential effects of two strains of Rhizophagus intraradices on dry biomass and essential oil yield and composition in Calamintha nepeta / Differential effects of two strains of Rhizophagus intraradices on dry biomass and essential oil yield and composition in Calamintha nepeta.

The aim of this work was to determine the effects of two geographically different strains of Rhizophagus intraradices (M3 and GA5) on the total biomass and essential oil (EO) yield and composition of Calamintha nepeta, with or without phosphorus (P) fertilization, under greenhouse conditions. The plant biomass was not significantly affected by any of the treatments, showing higher values in control plants. Strains had a differential response in their root colonization rates: M3 reduced these parameters while GA5 did not modify them. Both strains affected EO yield in absence of P fertilization: M3 promoted EO yield in C. nepeta plants and GA5 resulted in negative effects. The percentage composition of EO was not significantly modified by either strain or P fertilization. M3 strain could be a potential fungal bioinoculant for production and commercialization of C. nepeta in the aromatic plant market.

The PAMPA datasets: a metagenomic survey of microbial communities in Argentinean pampean soils.

BACKGROUND: Soil is among the most diverse and complex environments in the world. Soil microorganisms play an essential role in biogeochemical cycles and affect plant growth and crop production. However, our knowledge of the relationship between species-assemblies and soil ecosystem processes is still very limited. The aim of this study was to generate a comprehensive metagenomic survey to evaluate the effect of high-input agricultural practices on soil microbial communities. RESULTS: We collected soil samples from three different areas in the Argentinean Pampean region under three different types of land uses and two soil sources (bulk and rhizospheric). We extracted total DNA from all samples and also synthetized cDNA from rhizospheric samples. Using 454-FLX technology, we generated 112 16S ribosomal DNA and 14 16S ribosomal RNA amplicon libraries totaling 1.3 M reads and 36 shotgun metagenome libraries totaling 17.8 million reads (7.7 GB). Our preliminary results suggested that water availability could be the primary driver that defined microbial assemblages over land use and soil source. However, when water was not a limiting resource (annual precipitation >800 mm) land use was a primary driver. CONCLUSION: This was the first metagenomic study of soil conducted in Argentina and our datasets are among the few large soil datasets publicly available. The detailed analysis of these data will provide a step forward in our understanding of how soil microbiomes respond to high-input agricultural systems, and they will serve as a useful comparison with other soil metagenomic studies worldwide.

Differential effects of two strains of Rhizophagus intraradices on dry biomass and essential oil yield and composition in Calamintha nepeta. / Differential effects of two strains of Rhizophagus intraradices on dry biomass and essential oil yield and composition in Calamintha nepeta.

The aim of this work was to determine the effects of two geographically different strains of Rhizophagus intraradices (M3 and GA5) on the total biomass and essential oil (EO) yield and composition of Calamintha nepeta, with or without phosphorus (P) fertilization, under greenhouse conditions. The plant biomass was not significantly affected by any of the treatments, showing higher values in control plants. Strains had a differential response in their root colonization rates: M3 reduced these parameters while GA5 did not modify them. Both strains affected EO yield in absence of P fertilization: M3 promoted EO yield in C. nepeta plants and GA5 resulted in negative effects. The percentage composition of EO was not significantly modified by either strain or P fertilization. M3 strain could be a potential fungal bioinoculant for production and commercialization of C. nepeta in the aromatic plant market.

Continuous and long-term monoxenic culture of the arbuscular mycorrhizal fungus Gigaspora decipiens in root organ culture.

Establishment of arbuscular mycorrhizal (AM) germplasm collections is complex because of the obligate biotrophic nature of AM fungi. Only a few AM species are routinely maintained in monoxenic culture with Ri T-DNA transformed roots as host. Incorporation of new AM species into this culture system is important for molecular, physiological, and taxonomical studies. Here we report for the first time the successful monoxenic culture of Gigaspora decipiens (JA2 strain) with transformed carrot (Daucus carota) roots. In vitro cultures were established from field-collected spores; sub-culture of newly in vitro formed spores was established over five successive generations for a period of 6 y. Although initial culture of field-collected spores was difficult successive sub-cultures appeared to be adapted to the in vitro growing conditions. The JA2 strain of G. decipiens completed its life cycle while maintaining its morphological characteristics, stability, and propagule viability under the monoxenic conditions over several generations. This stable and homogeneous monoxenic material obtained for G. decipiens is part of the Banco de Glomeromycota In Vitro (BGIV, http://www.bgiv.com.ar), and could facilitate morphological, physiological, and molecular analysis of this AM species.