Tissue magnesium and calcium affect arbuscular mycorrhiza development and fungal reproduction

 Applications of high levels of MgSO₄ resulted in reduced root colonization and sporulation by Glomus sp. (INVAM isolate FL329) with sweet potato and onion in aeroponic and sand culture, respectively. Onion shoot-Mg concentrations were elevated when a nutrient solution containing 2.6 or 11.7 mm MgSO₄ was applied. Magnesium application depressed tissue-Ca levels. With lower Ca in the tissue, colonization was reduced from > 30% of root length to < 10%, and sporulation from > 1200 to ca. 200 spores per plant, 10 weeks after transplantation and the start of nutrient application. These effects on colonization and sporulation were independent of changes in tissue-P concentration. High Mg/low Ca tissue concentrations induced premature root senescence, which may have disrupted the mycorrhizal association. Our results confirm the importance of Ca for the maintenance of a functioning mycorrhiza. Accepted: 3 October 1997     

Heavy-Metal Stress and Developmental Patterns of Arbuscular Mycorrhizal Fungi

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices, had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices. A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.     

Increased Sporulation of Vesicular-Arbuscular Mycorrhizal Fungi by Manipulation of Nutrient Regimens

Adjustment of pot culture nutrient solutions increased root colonization and sporulation of vesicular-arbuscular mycorrhizal (VAM) fungi. Paspalum notatum Flugge and VAM fungi were grown in a sandy soil low in N and available P. Hoagland nutrient solution without P enhanced sporulation in soil and root colonization of Acaulospora longula, Scutellospora heterogama, Gigaspora margarita, and a wide range of other VAM fungi over levels produced by a tap water control or nutrient solutions containing P. However, Glomus intraradices produced significantly more spores in plant roots in the tap water control treatment. The effect of the nutrient solutions was not due solely to N nutrition, because the addition of NH₄NO₃ decreased both colonization and sporulation by G. margarita relative to levels produced by Hoagland solution without P.     

Differential host plant performance as a function of soil arbuscular mycorrhizal fungal communities: experimentally manipulating co-occurring Glomus species

In order to evaluate host plant performance relative to different soil arbuscular mycorrhizal fungal (AMF) communities, Andropogon gerardii seedlings were grown with nine different AMF communities. The communities consisted of 0, 10, or 20 spores of Glomus etunicatum and 0, 10, or 20 spores of Glomus intraradices in all possible combinations. Spores were produced by fungal cultures originating on A. gerardii in a serpentine plant community; seeds of A. gerardii were collected at the same site. The experiment was performed in the greenhouse using a mixture of sterilized serpentine soil and sand to which naturally occurring non-mycorrhizal microbes were added. There was no difference in root AMF colonization rates between single species communities of either G. etunicatum or G. intraradices, but G. intraradices enhanced plant growth and G. etunicatum did not. However, plants grew larger with some combinations of G.␣intraradices plus G. etunicatum than with the same quantity of G. intraradices alone. These results suggest the potential for niche complementarity in the mycorrhizal fungi. That G. etunicatum only increased plant growth in the presence of G. intraradices could be illustrative of why AMF that appear to be parasitic or benign when examined in isolation are maintained within multi-species mycorrhizal communities in nature.     

A new system using Solanum tuberosum for the co-cultivation of Glomus intraradices and its potential for mass producing spores of arbuscular mycorrhizal fungi

Freshly harvested potato tubers, Solanum tuberosum var ‘Pukhraj’, were inoculated for transformation with Agrobacterium rhizogenes strain Ri1600. Hairy roots were formed after 8 days of co-cultivation and the transformation efficiency was 40 %. The transformants were transferred from Murashige and Skoog medium (MS) to Modified White’s medium (MW) and finally on a hormone-free minimal medium (M). The putative transformants were confirmed using rolA and rolB gene specific primers for the polymerase chain reaction (PCR) analysis. The root inducing (Ri) T-DNA transformed potato roots were co-cultured with Glomus intraradices (CMCCROC7) to obtain arbuscular mycorrhizal root organ cultures (AM-ROC dual cultures), which were used for studying the symbiosis with Glomus intraradices and the potential for spore production in vitro. Sporulation was comparable with the existing in vitro carrot-dual culture system. Around 60,250 spores/jar could be harvested with around 38,314 extraradical spores/jar and around 21,936 intraradical spores/jar. The new method using potato is certainly promising for the mass production of mycorrhizal biofertilizers. The viability of the spores when tested on potato roots was nearly 100 % and more than half of the roots were colonized 12 weeks after inoculation.     

Improved aeroponic culture of inocula of arbuscular mycorrhizal fungi

We compared conventional atomizing disc aeroponic technology with the latest ultrasonic nebulizer technology for production of Glomus intraradices inocula. The piezo ceramic element technology used in the ultrasonic nebulizer employs high-frequency sound to nebulize nutrient solution into microdroplets 1 μm in diameter. Growth of pre-colonized arbuscular mycorrhizal (AM) roots of Sudan grass was achieved in both chambers used but both root growth and mycorrhization were significantly faster and more extensive in the ultrasonic nebulizer system than in the atomizing disc system. Shearing of the AM fungi (AMF) infected roots in both the systems did not reduce inoculum viability, as evident from the MPN data. However, sheared roots from the ultrasonic nebulizer system had significantly more infective propagules than those produced in the atomizing disc system. Thus, the latest ultra-sonic nebulizer aeroponic technology appears to be superior and an alternative to conventional atomizing disc or spray nozzle systems for the production of high-quality AMF inocula. These can be used in small doses to produce a large response, which is a prerequisite for commercialization of AMF technology. Accepted: 11 January 2000     

Dual axenic culture of sheared-root inocula of vesicular-arbuscular mycorrhizal fungi associated with tomato roots

Surface-sterilized sheared-root inocula of two vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus intraradices and G. versiforme) from pot cultures associated with excised tomato roots showed significant sporulation and the production of an extensive hyphal biomass. As many as 10²–10³ axenic mature spores were recovered in Petri dishes during 3 months incubation in the dark. Propagules of both species were able to complete their vegetative life cycle in vitro and efficiently colonize Acacia albida roots after 1 month under greenhouse conditions. The effectiveness of 0.5 cm pieces of VAM roots as starter inocula indicates the high inoculum potential of intravesicle propagules.     

Colonization of wheat roots from seed‐applied spores of Idriella (Microdochium) bolleyi: A biocontrol agent of take‐all

Wheat seedlings were grown in containers of perlite in a glasshouse, and spores of Idriella bolleyi were applied either to the seeds in alginate gel or to the perlite as aqueous suspensions. Growth and sporulation of the fungus on seeds and roots were assessed by plating methods and by retrieval of spores from water that drained from the plant containers. Idriella bolleyi sporulated heavily on seeds for up to 3 weeks when applied to them in alginate gel, andmost of the root system was infected from newly formed spores that were carried in percolating water. Removal of the inoculated seeds from seedlings at 7 days reduced the degree of root infection and temporarily reduced the number of spores in drainage water; however, spore numbers increased to control levels (seeds attached) by 21 days, indicating subsequent sporulation on the roots. When spores were applied to perlite containing young (0–3 days) seedlings I. bolleyi colonized the seeds, sporulated heavily on them and extensively colonized the roots. However, it established poorly on seeds and roots if added to the perlite when seedlings were 11 days or older. The results suggest that early growth on seeds is necessary for establishment of a high population of I. bolleyi, and that cycles of sporulation on seeds and then roots contribute to colonization of the rhizosphere. The features associated with the rhizosphere competence of I. bolleyi are compared with those for systemic colonization of the xylem by vascular wilt fungi, and suggest a new approach to the selection of root‐colonizing biocontrol agents.     

Colonization potential of in vitro-produced arbuscular mycorrhizal fungus spores compared with a root-segment inoculum from open pot culture

 A reliable inoculum, free from other microorganisms, to produce arbuscular mycorhizal (AM) plants is of the greatest importance when studying the interaction between AM plants and soil microorganisms. We investigated the colonization of leeks from monoxenic in vitro-produced Glomus intraradices spores. The isolated spores were produced using a two-compartment in vitro growth system previously described. A spore suspension was used as inoculum and was compared to the inoculum potential of endomycorrhizal root segments of pot-grown leek (Allium porrum L.) plants. The leeks were grown in a controlled environment and two types of sterilized growth media were tested: calcined montmorillonite clay and a soil mix. Root colonization progressed faster in the soil mix than in the clay. However, in this medium, after an initial delay, root colonization from in vitro-produced spores was essentially the same as that observed with the root-segment inoculum, reaching 44% and 58% respectively, after 16 weeks. Leek roots colonized by the monoxenically-produced spores harbored only the studied AMF fungi while the roots colonized from the root segments were substantially contaminated by other fungi. Accepted: 25 December 1998     

A comparison of AM fungi inoculants using Capsicum and Polianthes in marginal soil amended with organic matter

 Different types of nursery inocula formulations, namely mixed indigenous cultures and Glomus intraradices Schenck and Smith, were compared with commercially available inoculants of AM fungi in a pot experiment using two horticultural crops, Capsicum and Polianthes. Soil-based inocula and soil beads produced the highest response in both crops. Glomus intraradices resulted in the highest yield in both Polianthes (45% increase in spike length) and Capsicum (112% increase in fruit yield). Among the commercial inocula tested, only Mycorise enhanced spike length (33%) and fruit yield (11%) in the two hosts. Overall AM colonization was higher in Polianthes than in Capsicum. Sheared root inocula of G. intraradices resulted in high colonization (upto 68%) but the yield enhancement was lower than with soil-based formulations. The mixed indigenous culture produced the highest number of spores and propagules and commercial inocula the lowest. Accepted: 2 February 1998     

Monoxenic in vitro production and colonization potential of AM fungus Glomus intraradices

The paper reports the establishment of mycorrhizal infection of a non-mycorrhizal Ri-T-DNA transformed carrot root when co-cultured with a surface sterilized sweet potato root segment colonized by arbuscular mycorrhizal (AM) fungus G. intraradices on minimal M medium. Extensive fungal hyphal emergence from each cut end of the mycorrhizal sweet potato root piece was observed in one week old cultures. These hyphae caused infection on contacting the transformed-carrot- root segment and produced many hyphae and spores both inside and outside the zone of the root after 6 week of growth. Axenically produced fungal propagules proliferated on the surface of fresh minimal M medium when sub-cultured without any root segment. On repeated sub-culturing, these propagules did not lose their ability to grow and produced many juvenile small spore-like vesicles during the non-symbiotic phase. Although these spores were morphologically and anatomically similar to their soil borne counter parts, they were much smaller. When placed in the vicinity of a fresh hairy root on the minimal medium or a Sudan grass seedling in sand culture, the axenically produced AM fungal propagules caused root infection, but the infection characteristics were significantly different to the original culture in terms of shape (spherical vs oval) and size (20 microm vs 45 microm) of the intraradical vesicles, and absence of 'H' branches. Sudan grass seedlings inoculated with the axenically cultured fungus showed significantly (P < 0.05) higher dry weights plant'. When compared to the plants inoculated with sand cultures, the growth parameters and the percentage infection were not significantly different. However, when both sources of inocula were used together, a synergistic effect on plant growth as well as root infection was observed.     

Taxon-specific oligonucleotide primers for detection of Glomus etunicatum

The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus etunicatum MD107, MD127, TN101, and FL329 were amplified by polymerase chain reaction (PCR) using ITS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, and 613 bp, respectively) were cloned and sequenced. The similarity among ITS region sequences from MD107, MD127, and TN101 was 99%, whereas the sequence similarity between the ITS regions of these three DNAs and that from FL329 was 91%. The 5.8S rDNA sequences of all four G. etunicatum isolates were identical. In contrast, major dissimilarities in the corresponding rDNA sequence regions of other glomalean taxa were observed. Oligonucleotide sequences unique to G. etunicatum were tested for their specificity in PCR amplification of genomic DNA from spores of 55 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, five G. intraradices, three G. claroideum, 16 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The G. etunicatum isolates were from a broad range of geographic regions and soils. The oligonucleotide pair GETU1:GETU2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all G. etunicatum. This primer pair did not prime PCR when template consisted of DNA from any of the other glomalean fungi or any of the non-mycorrhizal controls, including roots of corn (Zea mays). In addition, the pair successfully detected G. etunicatum in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn roots using modified ITS1:ITS4 primers. In the phylogenetic analysis of Glomus 5.8S and ITS2 rDNA region sequences, which included 500 bootstrap data sets, confidence in the G. etunicatum branch was very strong (90%) and clearly independent of G. claroideum and G. intraradices, to which it is very closely related. Accepted: 15 October 2000     

Effects of single and mixed inoculation with two arbuscular mycorrhizal fungi in two different levels of phosphorus supply on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers

Aims

This study aimed to determine the effect of arbuscular mycorrhizal (AM) fungi and phosphorus (P) supply levels on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers.

Methods

Two commercial AM fungal isolates of Glomus intraradices (IFP Glintra) and Glomus mosseae (IFP Glm) which differ in their life cycles were used. Sweet potato plants were grown in a horizontal split-root system that consisted of two root compartments. A root-free fungal compartment that allowed the quantification of mycelial development was inserted into each root compartment. The two root compartments were inoculated either with the same or with different AM isolates, or remained free of mycorrhizal propagules. Each fungal treatment was carried out in two P supply levels.

Results

In the low P supply level, mycorrhizal colonization significantly increased β-carotene concentrations in sweet potato tubers compared with the non-mycorrhizal plants. Glomus intraradices appeared to be more efficient in increasing β-carotene concentrations than G. mosseae. Dual inoculation of the root system with the two mycorrhizal fungi did not result in a higher increase in tuber β-carotene concentrations than inoculation with the single isolates. Improved P nutrition led to higher plant tuber biomass but was not associated with increased β-carotene concentrations.

Conclusions

The results indicate a remarkable potential of mycorrhizal fungi to improve β-carotene concentrations in sweet potato tubers in low P fertilized soils. These results also suggest that β-carotene metabolism in sweet potato tubers might be specifically activated by root mycorrhizal colonization.     

Growth and nutrient uptake of sorghum cultivated with vesicular-arbuscular mycorrhiza isolates at varying pH

This study was conducted to determine the effects of different pH regimes on root colonization with four vesicular-arbuscular mycorrhiza (VAM) isolates, and VAM effects on host plant growth and nutrient uptake. Sorghum [Sorghum bicolor (L.) Moench] was grown at pH 4.0, 5.0, 6.0 and 7.0 (±0.1) in hydroponic sand culture with the VAM isolates Glomus etunicatum UT316 (isolate E), G. intraradices UT143 (isolate I), G. intraradices UT126 (isolate B), and an unknown Glomus isolate with no INVAM number (isolate A). Colonization of roots with the different VAM isolates varied differentially with pH. As pH increased, root colonization increased with isolates B and E, remained unchanged with isolate I, and was low at pH 4.0 and high at pH 5.0, 6.0, and 7.0 with isolate A. Isolates E and I were more effective than isolates A and B in promoting plant growth irrespective of pH. Root colonization with VAM appeared to be independent of dry matter yields or dry matter yield responsiveness (dry matter produced by VAM compared to nonmycorrhizal plants). Dry matter yield responsiveness values were higher in plants whose roots were colonized with isolates E and I than with isolates A and B. Shoot P concentrations were lower in plants colonized with isolates E and I than with isolates A and B or nonmycorrhizal plants. This was probably due to the dilution effect of the higher dry matter yields. Neither the VAM isolate nor pH had an effect on shoot Ca, Mg, Zn, Cu, and Mn concentrations, while the VAM isolate affected not only P but also S, K, and Fe concentrations. The pH x VAM interaction was significant for shoot K, Mg, and Cu concentrations.     

Arbuscular mycorrhiza colonization and development at suboptimal root zone temperature

Temperature has a strong influence on the activity of living organisms. This study, involving two indoor experiments, evaluated the effects of root zone temperature (10, 15 and 23°C) on the formation and development of arbuscular mycorrhizae (AM). In the first trial, greenhouse-grown sorghum [Sorghum bicolor (L.) Moench] was either colonized by Glomus intraradices Schenck & Smith or left non-mycorrhizal. Root length, root and shoot weight and root colonization were measured after 5, 10 and 15 weeks of plant growth. Although suboptimal root zone temperatures reduced growth in both mycorrhizal and non-mycorrhizal plants, mycorrhizal plants were larger than non-mycorrhizal plants after 15 weeks at 15 and 23°C. At suboptimal root zone temperatures, mycorrhizal inoculation sometimes slightly reduced root development. AM colonization was more affected than root growth at suboptimal root zone temperatures. Colonization was markedly reduced at 15°C compared with 23°C, and almost completely inhibited at 10°C. The second experiment was conducted in vitro using transformed carrot (Daucus carota L.) roots supporting G. intraradices. Mycelium length and spore number were measured weekly for 15 weeks. Spore metabolic activity (iodonitrotetrazolium reduction), root length and percentage root colonization were measured after 15 weeks. G. intraradices sporulation was reduced at temperatures below 23°C, while spore metabolic activity was significantly reduced only at 10°C. Root length and in particular percentage colonization were decreased at suboptimal temperatures. A negative interaction between AM hyphal growth and root growth resulting in reduced probability of contact at suboptimal root zone temperatures is proposed to explain the greater reduction observed in root colonization than in root and hyphal growth.     

Two Arbuscular Mycorrhizal Fungi Alleviates Drought Stress and Improves Plant Growth in Cinnamomum migao Seedlings

Cinnamomum migao plants often face different degrees of drought in karst habitats, which can lead to plants’ death, especially in the seedling stage. Widespread of arbuscular mycorrhizal (AM) fungi in karst soils have the potential to address this drought, which is a threat to C. migao seedlings. We inoculated C. migao seedlings with spores from Glomus lamellosum and Glomus etunicatum, two AM fungi widely distributed in karst soils, to observe seedling growth response after simulated drought. Our results showed that 40 g of G. lamellosum and G. etunicatum significantly promoted the growth of C. migao seedlings, 120 days after inoculation. Following a 15-day drought treatment, root colonization of the seedlings with G. lamellosum or G. etunicatum had lower the accumulation of malondialdehyde (MDA) and increased the accumulation of enzymes and osmotic substances in the seedlings. The relative water content in different organs (roots, stems, and leaves) of the drought-stressed seedlings was higher in plants with G. lamellosum or G. etunicatum than in plants without AM fungi colonization. Our results showed that inoculation with AM fungi was an effective means to improve the drought resistance of C. migao seedlings.     

Mycorrhizal fungi and nonhydraulic root signals of soil drying

We propose that mycorrhizal colonization of roots alters nonhydraulic root to shoot communication of soil drying. Split-root rose (Rosa hybrida L. cv Samantha) plants—one side of the root system colonized by Glomus intraradices Schenck & Smith, the other side nonmycorrhizal—displayed different stomatal conductances upon partial drying, depending upon whether mycorrhizal or nonmycorrhizal roots were dried. No differences in leaf water status were observed among control plants and those whose mycorrhizal or nonmycorrhizal roots were dried.     

Toxicity of diesel fuel to germination,growth and colonization of <Emphasis Type="Italic">Glomus intraradices</Emphasis> in soil and <Emphasis Type="Italic">in vitro</Emphasis> transformed carrot root cultures

Phytoremediation is the use of selected plants to decontaminate polluted environments. Arbuscular mycorrhizal fungi (AMF) may potentially be useful for phytoremediation, but it is not known how petroleum hydrocarbons influence AMF spore germination and hyphal growth. To address this question, germination of spores and germ tube growth of Glomus intraradices Schenck and Smith and Glomus aggregatum Schenck and Smith were assessed in soil contaminated with up to 3% (w/v) of F2 diesel oil or HAGO reference oil. Hyphal growth, colonization and progeny spore production were assessed in vitro using transformed root cultures of Daucus carota and G. intraradices spores in a F2 diesel contaminated medium. In addition, extraradical hyphal growth of G. intraradices colonizing Daucus carota in the presence of F2 diesel was studied. Neither F2 diesel nor HAGO reference oil affected spore germination or germ tube growth in soil. However, in the presence of plant roots, germ tube growth of G. intraradices was reduced and delayed in the presence of F2 diesel and root colonization was not detected. Hyphal growth of pre-colonized carrot roots by G. intraradices was reduced and delayed in F2 contaminated medium compared to controls. F2 diesel did not inhibit spore germination of these AMF species but did reduce colonization, germ tube and hyphal growth. These results suggest that AMF inoculum can be established in petroleum-contaminated sites. However, it may prove beneficial to plant pre-colonized plants to increase the probability of sufficient AMF colonization and growth. The likely mechanism(s) of petroleum toxicity in this plant-microbe system was discussed.     

AM 真菌影响三叶草根系抗氧化酶活性的系统效应

本文对三叶草接种AM 真菌根内球囊霉, 用盆栽试验和分根试验测定根系的菌根侵染率和抗氧化酶活性, 研究AM 真菌对根系抗氧化酶活性的影响以及该影响的系统性。结果表明, 盆栽试验中接种根内球囊霉显著提高了根系中SOD、POD、CAT 的活性, 表明AM 真菌可以促进根系的抗氧化酶活性; 分根试验中一半根系接种了根内球囊霉的植株, 其另一半未接种的根系SOD、POD 活性也增加, 表明AM 真菌对根系抗氧化酶系统的促进具有系统效应。由于抗氧化酶系统是植物产生抗逆性的生理生化基础, 可以推测, AM 真菌对根系抗氧化酶活性的系统性提高有助于保护根系整体, 而非仅仅保护受侵染根段。     

Effects of anthracene on development of an arbuscular mycorrhizal fungus and contribution of the symbiotic association to pollutant dissipation

The influence of anthracene, a low molecular weight polycyclic aromatic hydrocarbon (PAH), on chicory root colonization by Glomus intraradices and the effect of the root colonization on PAH degradation were investigated in vitro. The fungus presented a reduced development of extraradical mycelium and a decrease in sporulation, root colonization, and spore germination when exposed to anthracene. Mycorrhization improved the growth of the roots in the medium supplemented containing 140 mg l⁻¹ anthracene, suggesting a positive contribution of G. intraradices to the PAH tolerance of roots. Anthracene disappearance from the culture medium was quantified; results suggested that nonmycorrhizal chicory roots growing in vitro were able to contribute to anthracene dissipation, and in addition, that mycorrhization significantly enhanced anthracene dissipation. These monoxenic experiments demonstrated a positive contribution of the symbiotic association to anthracene dissipation in the absence of other microorganisms. In addition to anthracene dissipation, intracellular accumulation of anthracene was detected in lipid bodies of plant cells and fungal hyphae, indicating intracellular storage capacity of the pollutant by the roots and the mycorrhizal fungus.