Ri T-DNA Transformed Carrot Roots Research Articles

Lead uptake by the symbiotic Daucus carota L.-Glomus intraradices system and its effect on the morphology of extra- and intraradical fungal microstructures.

This work examines the strategies adopted by an arbuscular mycorrhizal symbiotic system to ameliorate environmental Pb stress by examining the concentrations of P, Fe, and Pb in the fungal microstructures and the host's root. In vitro cultures of Ri-T DNA-transformed carrot (Daucus carota L.) roots were inoculated with Glomus intraradices and treated with Pb(NO3)2 solution and the extraradical spores and mycelia (S/M) and the root with the vesicles, mycelia, and root cells were subsequently analyzed by polarized energy dispersive x-ray fluorescence (PEDXRF) spectrometry. Upon Pb treatment, within the root, the percentages of mycorrhizal colonization, the vesicles, and mycelia increased as well as the areas of the vesicles and the (extraradical) spores, although the number of spores and arbuscules decreased. The S/M and the mycorrhizal root showed enhanced concentrations of Pb, Fe, and P. These were particularly marked for Fe in the Pb-treated cultures. This indicates a synergistic relationship between the arbuscular mycorrhizal fungus and the host that confers a higher Pb tolerance to the latter by the induction of higher Fe absorption in the host. The intraradical vesicle, mycelia, and arbuscule numbers are interpreted as a "tactic to divert" the intraradical Pb traffic away from the root cells to the higher affinity cell walls of the arbuscular mycorrhizal fungi (AMF) microstructures in the apoplast. The results of this work show that the symbiosis between the AMF G. intraradices and the host plant D. carota distinctly improves the latter's Pb tolerance, and imply that the appropriate metal tolerant host-AMF combinations could be employed in process designs for the phytoremediation of Pb.

The sterol biosynthesis inhibitor molecule fenhexamid impacts the vegetative compatibility of Glomus clarum

The vegetative compatibility of the arbuscular mycorrhizal fungus (AMF) Glomus clarum MUCL 46238 was evaluated after continuous exposure to fenhexamid, a sterol biosynthesis inhibitor (SBI). Three lineages of this AMF were cultured in vitro for five generations in association with Ri T-DNA transformed carrot roots in the presence of 0, 5 or 10mgl(-1) of fenhexamid. Whatever the AMF generation, fenhexamid at 5 and 10mgl(-1) had no significant impact on the number of spores produced. However, vegetative compatibility tests (VCT) conducted with spores from the three lineages, in the presence of 10mgl(-1) of fenhexamid, impacted the anastomosis process. At this concentration, the morphology of the germ tubes was modified. In addition, nitrotetrazolium-trypan blue staining revealed that 10mgl(-l) of fenhexamid significantly reduced the probability of fusion between the germ tubes regardless of the culture conditions (i.e. absence or presence of fenhexamid) preceding the VCT. Our results demonstrated that spore production was not affected by fenhexamid, while anastomosis between germ tubes was decreased. This suggested that high concentrations, accumulation or repeated application of this SBI fungicide may impact the community structure of AMF in soil.

Detection of a novel bacterium associated with spores of the arbuscular mycorrhizal fungusGigaspora margarita

With PCR-denaturing gradient gel electrophoresis analysis, two bacterial 16S rRNA gene V3 region sequences, 7A and 7B, were detected in association with the crushed spores of the arbuscular mycorrhizal fungus Gigaspora margarita W.N. Becker & I.R. Hall 1976 MAFF520054. DNA sequencing and phylogenetic analysis revealed that 7B was mostly related to the documented cytoplasm endosymbiotic bacterium Candidatus Glomeribacter gigasporarum of G. margarita, but 7A could not be confidently assigned to a known taxon. Further characterization of 7A was conducted by obtaining its almost complete 16S rRNA gene sequence via PCR amplification and sequencing. BLAST search indicates that the 16S rRNA gene sequence did not match any identified species sequences in the GenBank database. Further detection revealed that 7A was also associated with the clean G. margarita MAFF520054 spores that were obtained by the surface-sterilized method or dual culture with Ri T-DNA transformed carrot roots. Many ellipse-shaped or egg-shaped bacterium-like organisms were clustered in layer 3 of the fungal spore wall by transmission electron microscopy observation. Our results indicate that 7A represents a novel bacterial population associated with G. margarita MAFF520054 spores, and its doubtless location (wall or cytoplasm) remains unclear based on the present data.

Morphological, ontogenetic and molecular characterization of Scutellospora reticulata (Glomeromycota)

The arbuscular mycorrhizal (AM) fungus Scutellospora reticulata (CNPAB11) was characterized using morphological, ontogenetic and molecular approaches. Spore ontogenesis was studied using Ri T-DNA transformed carrot roots and observations were compared with those published for eight other, pot-cultured, Scutellospora species. The sporogenesis of S. reticulata exhibited an unreported pattern of outer spore wall differentiation. In addition, Denaturing Gradient Gel Electrophoresis (DGGE), targeting the V9 region of the SSU nrDNA, was used to differentiate S. reticulata from 16 other Scutellospora species and results were confirmed by sequencing analysis. Phylogenetic analyses, using nearly full length SSU nrDNA sequences, grouped S. reticulata in a cluster together with S. cerradensis and S. heterogama, species that share similar spore wall organization and also possess ornamented external walls. PCR-DGGE and sequence analysis revealed intragenomic SSU nrDNA polymorphisms in four out of six Scutellospora species tested, and demonstrated that SSU nrDNA intragenomic polymorphism could be used as a marker to differentiate several closely related Scutellospora species.

Isolated root caps, border cells, and mucilage from host roots stimulate hyphal branching of the arbuscular mycorrhizal fungus, Gigaspora gigantea

Unlike previous reports that have shown that water soluble and volatile compounds from roots or root exudates play an important role in precolonization events during arbuscular mycorrhizal (AM) fungus-host root interactions (Bécard & Piché 1989, Giovannetti et al. 1993), the results shown here deal with particulate and viscous fractions isolated from host roots. Root caps and a slow sedimenting particulate fraction (SSPF) were rapidly isolated and separated from Ri T-DNA transformed carrot roots (D. carota) grown in liquid culture. In addition, border cells (BC) and mucilage were isolated from aseptically grown corn seedlings (Zea mays). Root caps, SSPF (composed mainly of small root cap fragments and some BCs), BCs, and mucilage all had an associated AM fungus hyphal branching stimulator. Root caps stored for 5 d at 4 degrees C appeared to either synthesize or slowly release the branching stimulator. Also, isolated root caps from roots grown in the absence of P contained more branch stimulating activity than those isolated from roots grown in the presence of P. Although the branching stimulation activity in particulate fractions was low compared to that of the exudate, the particulate fractions can stick to the root surface at considerable distances from the root tip. This may be significant during the infection and colonization of host roots at sites far removed from the primary location of exudation.

Development of extraradical mycelium of Scutellospora reticulata under root-organ culture: spore production and function of auxiliary cells

The development of the extraradical mycelium and auxiliary cells and spore production of Scutellospora reticulata in association with Ri T-DNA transformed carrot roots was followed under root-organ culture conditions. Extraradical mycelium development followed classical lag-exponential-plateau phases, with an additional late decline phase in number of auxiliary cells. Spore production started in parallel with a critical extraradical mycelium biomass produced, continued long after root growth ceased and during the late decline in auxiliary cells number. Isolated auxiliary cells were shown to exhibit hyphal re-growth, but not root colonization, either in situ or in vitro. These results showed that root and extraradical mycelium development were intimately associated in a sequence where both grew together during active root growth, followed during root aging by a period in which only the fungus developed. Spore production appeared dependent on a critical extraradical mycelium biomass and on the re-allocation of resources from both the intraradical mycelium and the auxiliary cells via the hyphal network.

Mycelium Development and Architecture, and Spore Production of Scutellospora reticulata in Monoxenic Culture with Ri T-DNA Transformed Carrot Roots

Mycelium Development and Architecture, and Spore Production of Scutellospora reticulata in Monoxenic Culture with Ri T-DNA Transformed Carrot Roots

Mycelium development and architecture, and spore production of Scutellospora reticulata in monoxenic culture with Ri T-DNA transformed carrot roots

Mycelium development and architecture and spore production were studied in Scutellospora reticulata from single-spore isolates grown with Ri T-DNA transformed carrot root-organ culture in monoxenic system. Culture establishment, anastomosis occurrence and auxiliary cell development also were examined. Seventy percent of the pregerminated disinfected spores colonized the transformed carrot roots. After 8 mo, the average spore production was 56 (24–130) per 30 cm3 of medium. Of the spores produced, 75% germinated and produced new generations in monoxenic culture. The mycelium network was formed by thick light-brown hyphae, which exhibit two major architecture patterns related to either root colonization or resource exploitation, and lower-order hyphae, bearing auxiliary cells, branched absorbing structures (BAS), hyphal swellings (HS) and forming anastomoses. BAS were formed abundantly in extramatrical mycelium and frequently had HS resembling vesicles, a feature not previously reported in the Gigasporaceae, to the best of our knowledge. Few anastomosis were observed within the mycelium and most often corresponded to a healing mechanism that form hypha bridges to reconnect broken hyphae or overcoming obstructed areas within a hypha. Numerous auxiliary cells were produced during culture development and their role was inferred.

Development of Acaulospora rehmii spore and hyphal swellings under root-organ culture

A strain of Acaulospora rehmii was, for the first time, successfully grown in vitro on Ri T-DNA transformed carrot roots allowing the in situ observation of Acaulospora spore development and of extraradical thin-walled hyphal swellings. The sporogenous hypha developed intercalarly along thin-walled coenocytic hyphae. The distal part of the sporogenous hypha swelled slightly as a sporiferous saccule primordium followed by the differentiation of a lateral spore primordium along the neck of the sporogenous hypha. Both structures matured simultaneously, and the sporiferous saccule began to collapse after spore maturation and complete differentiation of the spore wall. Several of the in situ observations on in vitro differentiated A. rehmii spores are concordant with previous ontogenic studies done on other Acaulospora species obtained from in vivo cultures. New and original observations on the early developmental stages of sporiferous saccules and spores and on the occurrence of small diameter intercalary hyphal swellings provide additional elements in the study of Acaulospora sporulation process and life cycle.

Intra- and intersporal diversity of ITS rDNA sequences in Glomus intraradices assessed by cloning and sequencing, and by SSCP analysis

The level of molecular variability in the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was assessed. We established several monoxenically growing single-spore cultures of the G. intraradices collected from a long-term soil tillage experiment in Tänikon (Switzerland). They were grown in symbiosis with Ri T-DNA transformed carrot roots. The ITS region was amplified from genomic DNA of fungal spores using ITS1 and ITS4 primers. The amplified ITS region of three of the fungal isolates was cloned and sequenced. All sequences obtained from our samples closely resembled those previously reported for G. intraradices, and no evidence for genetic exchange was found on the interspecific or intergeneric level. Phylogenetic analysis shøwed the presence of at least two different sequence families within a single-spore isolate. The single strand conformation polymorphism (SSCP) method detected different sequence types in a single PCR sample. This technique enabled us to detect several distinct ITS sequences within each of the single-spore isolates of the AMF. Estimates of intra- and intersporal sequence diversity were obtained by comparing diversity within and among the single-spore isolates. A higher level of diversity was found among spores than within a single spore.

In vitro co-culture of two AMF isolates Gigaspora margarita and Glomus intraradices on Ri T-DNA transformed roots

An in vitro system using Ri T-DNA transformed carrot roots as the host bearing simultaneously different arbuscular mycorrhizal (AM) fungi belonging to two genera Glomus intraradices (Smith and Schenck) and Gigaspora margarita (Becker and Hall) was developed. Co-existence appeared healthy and harmonious, as both the generic species showed extensive hyphal proliferation and sporulation. The co-culture model under the in vitro system appears especially appropriate for further investigations on the competition and on the interaction mechanism involved in such types of associations occurring in nature and also as a model approach towards mass production of multiple mycorrhizal fungal isolates. This is a first report of successful co-culture of two genera of AM fungi under in vitro conditions.

Intraradical sporulation of AM Gigaspora margarita in long-term axenic cultivation in Ri T-DNA carrot root

Arbuscular mycorrhizal (AM) fungi are obligate biotrophic organisms. Root organ culture (ROC) can be used to grow these fungi under in vitro conditions. The ROC technique was used with Gigaspora margarita and Ri T-DNA transformed carrot roots to examine the fungal growth and physiology under long-term axenic symbiosis. The fungus formed spores inside the host roots under in vitro conditions. Sporulation was a temporal phenomenon found in dual cultures more than 18–20 mo old. The spores were formed singly or in rare cases clusters of two or three. No preferential zone of formation was found. The spores formed intraradically were 10–15% of the total spores formed in a single culture. These intraradical spores were analysed for their morphology and DNA polymorphism pattern with spores which had formed conventionally in the medium. Both analyses showed no detectable variation. This is the first report of spore formation by Gigaspora inside the roots of a host.

Arbuscular mycorrhizal phosphate transport under monoxenic conditions using radio-labelled inorganic and organic phosphate.

Compartmented monoxenic cultures of Ri T-DNA transformed carrot roots and a symbiotic arbuscular mycorrhizal fungus demonstrated for the first time that phosphate in an organic form (32P-labelled AMP) may be hydrolysed by extra-radical mycorrhizal hyphae in the absence of other organisms, and subsequently utilized as a mineral nutrient source by the host plant after fungal transport.

In vitro propagation and life cycle of the arbuscular mycorrhizal fungus Glomus etunicatum

Progress in understanding the biology of arbuscular mycorrhizal fungi is hampered by the limited number of species that can be successfully propagated and studied in vitro. We report the establishment of monoxenic cultures of Glomus etunicatum in association with excised Ri T-DNA transformed carrot roots. The fungus can be propagated in vitro using monoxenically formed resting spores and/or colonized root fragments. Modified White's medium buffered with 10 mM MES (pH 6) or MOPSO (pH 6.5) was most optimal for the host root growth as well as for G. etunicatum spore germination and mycorrhiza formation. The number of resting spores formed in vitro correlated positively with the length of roots occupied by arbuscular mycorrhizal structures, including arbuscules and vesicles. Spores first appeared in dual cultures within two weeks of root inoculation. Sporulation was asynchronous and continued until root senescence. Under applied culture conditions, spores achieved mature appearance within 5–7 d after their initiation. Approximately 6% of monoxenic spores were aborted at different stages of their development. Although G. etunicatum spores formed in vitro exhibited general morphological and anatomical similarity to soil-borne inoculum, they were significantly smaller and had thicker spore walls than their soil-borne counterparts. Caution should, therefore, be exercised in utilizing the in vitro system as a model of growth and development of glomalean fungi in soil.

In vitro mass-production of the arbuscular mycorrhizal fungus, Glomus versiforme, associated with Ri T-DNA transformed carrot roots

The rate of in vitro spore formation of the arbuscular mycorrhizal fungus Glomus versiforme was followed in Petri dishes, using mycorrhizal root-segment inoculum associated with Ri T-DNA transformed carrot roots. Three phases of sporulation were observed: a lag phase, a period of intensive spore production and a plateau phase. An average of 9500 spores per Petri dish was produced after 5 months of dual culture. The root-organ culture system supported extensive root colonization, with many arbuscules and vesicles being formed. The fungus, both within root-segments and as spores produced, was viable and able to complete its life cycle in vitro. The mycorrhizal root-segments, however, exhibited higher inoculum potential due to the numerous vesicles and extensive intraradical mycelium.

Some studies relating to "independent" growth of vesicular–arbuscular endophytes

In dual culture with Ri T-DNA transformed carrot roots nutrient conditions were sought that would totally suppress mycorrhizal infection while stimulating "independent" growth of vesicular–arbuscular endophytes. In this system fungal development in the agar was comparable in extent with that normally occurring only after mycorrhizal infection has become well established; it differed qualitatively from that characteristic of germinating spores and extramatrical mycelium in such cultures. There was profuse development of arbuscule-like branches, many anastomoses, and increased branching of extension hyphae. Biomass increased up to fivefold when the mycelium was detached in situ from the inoculum (root pieces or spores). Growth from explants was little more than residual and was hampered by extensive bacterial contamination at the desired pH, near neutrality. The rationale of this approach to the culture of vesicular–arbuscular endophytes is discussed.