Arbuscular Mycorrhizal Fungal Community Composition in Carludovica palmata, Costus scaber and Euterpe precatoria from Weathered Oil Ponds in the Ecuadorian Amazon

Arbuscular mycorrhizal fungi (AMF) are ubiquitous to most natural and anthropized ecosystems, and are often found in polluted environments. However, their occurrence and community composition in highly weathered petroleum-polluted soils has been infrequently reported. In the present study, two ponds of weathered crude oil and their surrounding soil from the Charapa field in the Amazon region of Ecuador were selected and root colonization by AMF of their native plants investigated. The AMF community was further analyzed in three selected plant species (i.e., Carludovica palmata, Costus scaber and Euterpe precatoria) present in the two ponds and the surrounding soil. A fragment covering partial SSU, the whole ITS and partial LSU rDNA region was amplified (i.e., 1.5 kb), cloned and sequenced from the roots of each host species. AMF root colonization exceeded 56% in all plant species examined and no significant difference was observed between sites or plants. For AMF community analysis, a total of 138 AMF sequences were obtained and sorted into 32 OTUs based on clustering (threshold ≥97%) by OPTSIL. The found OTUs belonged to the genera Rhizophagus (22%), Glomus (31%), Acaulospora (25%) and Archaeospora (22%). Glomus and Archaeospora were always present regardless of the plant species or the site. Acaulospora was found in the three plant species and in the two ponds while Rhizophagus was revealed only in the surrounding soil in one plant species (Euterpe precatoria). Our study contributed to the molecular community composition of AMF and revealed an unexpected high presence of four AMF genera which have established a symbiosis with roots of native plants from the Amazon forest under high polluted soil conditions

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. © 2011 Springer-Verlag

Cryopreservation of in vitro-produced Rhizophagus species has minor effects on their morphology, physiology, and genetic stability

Cryogenic storage is considered to be the most convenient method to maintain phenotypic and genetic stability of organisms. A cryopreservation technique based on encapsulation-drying of in vitro-produced arbuscular mycorrhizal fungi has been developed at the Glomeromycota In Vitro Collection. In this study, we investigated fungal morphology (i.e., number and size of spores, number of branched absorbing structures (BAS), hyphal length, and number of anastomosis per hyphal length), activity of acid phosphatase and alkaline phosphatase in extraradical hyphae, and variation in amplified fragment length polymorphism (AFLP) profiles of in vitro-produced isolates of five Rhizophagus species maintained by cryopreservation for 6 months at -130 °C and compared to the same isolates preserved at 27 °C. Isolates were stable after 6 months cryopreservation. Comparing isolates, the number of BAS increased significantly in one isolate, and hyphal length decreased significantly in another isolate. No other morphological variable was impacted by the mode of preservation. Phosphatase activities in extraradical hyphae and AFLP profiles were not influenced by cryopreservation. These findings indicate that cryopreservation at -130 °C of encapsulated-dried and in vitro-produced Rhizophagus isolates (i.e., Rhizophagus irregularis, Rhizophagus fasciculatus, Rhizophagus diaphanous, and two undefined isolates) is a suitable alternative for their long-term preservation. © 2013 Springer-Verlag Berlin Heidelberg

Trichoderma harzianum elicits defence response genes in roots of potato plantlets challenged by Rhizoctonia solani

Biological control of Rhizoctonia solani with Trichoderma harzianum has been demonstrated in several studies. However, none have reported the dynamics of expression of defence response genes. Here we investigated the expression of these genes in potato roots challenged by R. solani in the presence/absence of T. harzianum Rifai MUCL 29707. Analysis of gene expression revealed an induction of PR1 at 168 h post-inoculation (hpi) and PAL at 96 hpi in the plants inoculated with T. harzianum Rifai MUCL 29707, an induction of PR1, PR2 and PAL at 48 hpi in the plants inoculated with R. solani and an induction of Lox at 24 hpi and PR1, PR2, PAL and GST1 at 72 hpi in the plants inoculated with both organisms. These results suggest that in the presence of T. harzianum Rifai MUCL 29707, the expression of Lox and GST1 genes are primed in potato plantlets infected with R. solani at an early stage of infection

Do arbuscular mycorrhizal fungi with contrasting life-history strategies differ in their responses to repeated defoliation ?

Arbuscular mycorrhizal (AM) fungi obligatorily depend on carbon (C) resources provided via the plant and therefore fluctuations in C availability may strongly and differently affect AM fungi with different life-history strategies (LHS). In the present study, we examined the effect of repeated defoliation of in vitro grown barrel medic (Medicago truncatula) on the spore and auxiliary cell (AC) production dynamics of a presumed r-strategist (Glomus intraradices) and a presumed K-strategist (Dentiscutata reticulata). Glomus intraradices modulated the production of spores directly to C availability, showing direct investment in reproduction as expected for r-strategists. In contrast, AC production of D. reticulata was not affected after a single defoliation and thus showed higher resistance to fluctuating C levels, as expected for K-strategists. Our results demonstrate that plant defoliation affects the production of extraradical C storage structures of G. intraradices and D. reticulata differently. Our results contribute towards revealing differences in LHS among AM fungal species, a step further towards understanding their community dynamics in natural ecosystems and agroenvironments

Cryopreservation of arbuscular mycorrhizal fungi from root organ and plant cultures

Long-term maintenance of arbuscular mycorrhizal fungi (AMF) by in vitro or in vivo subcultivation is often expensive and time-consuming and could present the risk of contaminations and possibly morphological, physiological, and genetic variations over time. Recently, in vitro produced AMF isolates belonging to the genus Rhizophagus were successfully cryopreserved at -130 °C following encapsulation-drying. Here, this method was tested on 12 single species cultures belonging to six different genera (i.e., Rhizophagus, Glomus, Claroideoglomus, Septoglomus, Paraglomus, and Gigaspora) produced in vitro or in vivo. Their viability was estimated, after 1 month of cryopreservation at -130 °C, by the percentage of potentially infective beads (i.e., the percentage of beads that contained at least one germinated propagule) for the in vitro produced species and the percentage of infective beads (i.e., the percentage of beads that contained at least one propagule able to colonize a new host plant in pot culture) for the in vivo produced species. With the exception of Gigaspora sp. MUCL 52331 and Septoglomus constrictus PER 7.2, no significant differences were observed in the viability of the single species cultures before and after cryopreservation. These results, thus, demonstrated the suitability of the cryopreservation method by encapsulation-drying for AMF species belonging to different genera and produced in vitro or in vivo. This method opens the door to the long-term preservation at ultra-low temperature of a large number of AMF species and for the preservation of species that are still recalcitrant to in vitro cultivation. © 2013 Springer-Verlag Berlin Heidelberg

Preservation at ultra-low temperature of in vitro cultured arbuscular mycorrhizal fungi via encapsulation-drying

At present, over 300 species of arbuscular mycorrhizal fungi (AMF) have been identified, most of which being stored in international collections. Their maintenance is mostly achieved in greenhouse via continuous culture on trap plants or in vitro in association with excised root organs. Both methods are work-intensive and for the former present the risk of unwanted contaminations. The in vitro root organ culture of AMF has become an alternative preventing contamination. Nevertheless, the risk for somaclonal variation during the sub-cultivation process cannot be excluded. A method for the long-term conservation that guarantees the stability of the biological material is thus highly demanded to preserve the microorganisms and their genetic stability. Here, 12 AMF isolates cultured in vitro in association with excised carrot roots were encapsulated in alginate beads and subsequently cryopreserved. Several protocols were tested taking into consideration culture age, alginate bead pre-drying, and rate of decrease in temperature. The viability of the AMF isolates was estimated by the percentage of potentially infective beads (%PIB) that measure the % of beads that contain at least one germinated propagule. Thermal behaviour of alginate beads was analysed by a differential thermal calorimeter before and after drying to estimate the frozen and unfrozen water during the cryopreservation process. It was shown that the spore damage was directly related to ice formation during cryopreservation. The encapsulation and culture age were also determinant parameters for the successful cryopreservation. Irrespective of the AMF isolate, the optimal procedure for cryopreservation comprised five steps: (1) the encapsulation of propagules (i.e. spores and mycorrhizal root pieces) isolated from 5 m old cultures, (2) the incubation overnight in trehalose (0.5 M), (3) the drying during 48 h at 27 °C, (4) the cryopreservation in the freezer at -130 °C following a two-step decrease in temperature: a fast decrease (∼12 °C min -1) from room temperature (+20 °C) to -110 °C followed by a slow decrease in temperature (∼1 °C min -1) from -110 °C to -130 °C, and (5) the direct thawing in a water bath (+35 °C). The % PIB was above 70 % for all the isolates and even above 95 % for 11 out of the 12 isolates after several months of storage at ultra-low temperature. All the isolates kept their capacity to associate to an excised carrot root in vitro and to reproduce the fungal life cycle with the production of several hundreds to thousands of spores after 2 m. This method opens the door for the long-term maintenance at ultra-low temperature of AMF isolates within international repositories. © 2012 The British Mycological Society

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

Modelling the sporulation dynamics of arbuscular mycorrhizal fungi in monoxenic culture

Spore production of arbuscular mycorrhizal fungi is important in inoculum production, and monoxenic culture is a promising way to produce large amounts of contaminant-free inoculum. Mass production of spores is therefore essential and mathematical models useful as descriptive and predictive tools of sporulation dynamics. We followed the sporulation dynamics of three Glomus strains i.e. G. intraradices, G. proliferum and G. caledonium, cultured monoxenically on a nutrient agar medium containing macro- and microelements, vitamins and sucrose. Three models (Schnute, logistic, and Gompertz) were fitted to the data and compared in order to select the most adequate model. The Schnute, model was the reference against which the two other models were tested. For all three Glomus strains examined, the sporulation dynamics followed a sigmoidal curve with a lag, a log, and a plateau phase. Visually, all three models fitted the data very well, with R-2 values ranging from 0.9703 to 0.9995. They thus appeared adequate for describing the temporal dynamics of spore production. In most cases the Gompertz model described sporulation as accurately as the Schnute model, but the performance of the logistic model was seldom as good. The Gompertz model is thus convenient for modelling the sporulation dynamics of Glomus strains grown in a well-defined nutrient agar medium. As such, its use may facilitate and help improve exploitation of monoxenic culture systems